Marie Lopez

Julie Gilbert

Jean Contreras

Ludovic Halby

Paola B Arimondo
email: paola.arimondo@cnrs.fr

Inhibitors of DNA Methylation

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How to inhibit DNA methyltransferases

The DNA methylation mechanism involves three main actors: DNA, DNA methyltransferase (DNMT) and the co-factor S-adenosyl-L-methionine (SAM). DNMTs scan DNA, recognize CpG sites and flip out 2'-deoxycytidine into the catalytic pocket of the enzyme. A cysteine residue (C1226 in human DNMT1, C711 in human DNMT3A and C652 in human DNMT3B) binds to position 6 of the cytosine DNMTs can be inhibited by targeting different parts of the catalytic pocket (the deoxycytidine binding site, the SAM binding site or both), by targeting allosteric sites or by interfering with their binding to DNA. When the DNA is the target, for example by DNA binders, it is important to design specific compounds that do not inhibit other enzymes acting on DNA. A possibility is to use DNA ligands specific of CpGs sites. An interesting alternative are compounds that recognize the cytidine binding site into the DNMT catalytic pocket and, in addition, bear a chemical moiety able to react with the catalytic cysteine. Cytidine analogs, leading to a suicidal convalent complex targeting, are the best example and are described in 17.2. Another strategy is to target the co-factor binding pocket by SAM analogs for example. Since SAM is the most widely used enzyme cofactor after ATP [START_REF] Struck | S-Adenosyl-Methionine-Dependent Methyltransferases: Highly Versatile Enzymes in Biocatalysis, Biosynthesis and Other Biotechnological Applications[END_REF], a challenge lies in the design of compounds specific for the DNMT SAM-binding site and not binding to other methyltransferases. The design of SAM analogs that also bind to the 2'-deoxycytidine binding site, such as bisubstrate inhibitors (Bon, 2021), could confer this specificity and in addition it could potentially give higher affinity compounds and more potent inhibitors. Below, we listed the principal inhibitors representing the different possible mechanisms of inhibition of DNMTs.

Chemistry and structure of DNMT inhibitors

DNMT inhibitors (DNMTi) are grouped in two families: the nucleoside inhibitors and the nonnucleoside ones (Figure 17.2). To date, two compounds, 5-azacytidine (5aza, azacytidine, Vidaza ® ) and 5-aza-2'-deoxycytidine (5azadC, decitabine, Dacogen™ and in combination Inqovi ® ) (Figure 17.2), are approved by the FDA for the treatment of hematological cancers. 5Aza and 5azadC were the first DNMTi and their story began with their synthesis in 1964 [START_REF] Sorm | 5-Azacytidine, a new, highly effective cancerostatic[END_REF]. They were initially tested as anti-metabolite agents against acute myelogenous leukemia (AML) [START_REF] Cihak | Biological effects of 5-azacytidine in eukaryotes[END_REF][START_REF] Sorm | 5-Azacytidine, a new, highly effective cancerostatic[END_REF], showing anticancer properties [START_REF] Evans | The in vivo activity of combinations of 5-azacytidine and cytidine on leukemia L-1210[END_REF]). However, it was in the late 70'searly 80's that a big turn was taken in their use when Jones and Taylor showed that non-toxic doses of 5aza and 5azadC induced cell differentiation by DNMT inhibition [START_REF] Jones | Cellular differentiation, cytidine analogs and DNA methylation[END_REF][START_REF] Taylor | Multiple new phenotypes induced in 10T12 and 3T3 cells treated with 5azacytidine[END_REF].

Figure 17.2. Selection of compounds described as DNMT inhibitors

NUCLEOSIDE ANALOGUES

NON-NUCLEOSIDE ANALOGUES FROM NATURAL SOURCES

A new paradigm was found, as these compounds were shown to be able to reprogram cells, i.e. induce silenced-genes expression and thus restore normal cell functions. The aza-nucleosides, once phosphorylated in cells and incorporated into DNA, replace the cytosine in the catalytic pocket of the enzyme and act as suicide substrates of DNMTs (cf. Figure 17.1). In DNA, at the CpG sites, 5azadC is flipped out from the double-helix into the catalytic pocket, the catalytic cysteine binds covalently to C6 position, but the β-elimination, essential to release the enzyme, cannot occur after the methyl group transfer because of the presence of a nitrogen atom at the C5 position. The enzyme is thus irreversibly trapped on the DNA and further degraded by the proteasome [START_REF] Santi | Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine[END_REF], inducing DNA demethylation.

Based on these findings, 5aza (Vidaza®) and 5azadC (Dacogen™) were approved in 2004 and 2006, respectively, by the FDA for the treatment of acute myelodysplastic syndrome (MDS), and chronic myelomonocytic leukemia (CMML). However, these drugs are chemically and metabolically unstable, present a low bioavailability [START_REF] Chan | 5-Azacytidine Hydrolysis Kinetics Measured by High-Pressure Liquid Chromatography and 13C-NMR Spectroscopy[END_REF][START_REF] Notari | Kinetics and mechanisms of degradation of the antileukemic agent 5azacytidine in aqueous solutions[END_REF], and, as they are incorporated at every deoxycytidine, are not selective [START_REF] Karahoca | Pharmacokinetic and pharmacodynamic analysis of 5-aza-2'deoxycytidine (decitabine) in the design of its dose-schedule for cancer therapy[END_REF]. Nevertheless, the proof of concept for DNMTi as a therapeutic target was established and since then many efforts have been dedicated to identify novel DNMT inhibitors (DNMTi).

First, the chemical instability of 5aza and 5azadC was addressed. Indeed, The 5-azacytosine moiety was

shown to be sensitive to rapid hydrolysis under mild condition over two steps; a reversible ring opening to the N-formylguanylribosylurea followed by an irreversible formation of guanylribosylurea specie [START_REF] Chan | 5-Azacytidine Hydrolysis Kinetics Measured by High-Pressure Liquid Chromatography and 13C-NMR Spectroscopy[END_REF]. More stable analogs were designed, like 5,6-dihydro-5-azacytidine, 2'-deoxy-5,6-dihydro-5azacytidine, 2'-deoxy-5-fluorocytidine and zebularine (cf. Figure 17.2). 5,6-Dihydro-5-azacytidine showed weak inhibition of DNA methylation and was withdrawn from clinical trials (Yogelzang, 1997). Its analog, 2'-deoxy-5,6-dihydro-5-azacytidine, was shown not to bind covalently to DNMTs, but simply to occupy the cytidine pocket [START_REF] Sheikhnejad | Mechanism of inhibition of DNA (cytosine C5)-methyltransferases by oligodeoxyribonucleotides containing 5,6dihydro-5-azacytosine[END_REF], resulting in methylation inhibition in several cell lines (CRF-CEM and HL60) with very low cell toxicity [START_REF] Matoušová | 2′-deoxy-5,6-dihydro-5-azacytidine-a less toxic alternative of 2′-deoxy-5-azacytidine: A comparative study of hypomethylating potential[END_REF]. 2'-Deoxy-5fluorocytidine was described as more stable in aqueous media and as potent in vitro, but it is currently rather recognized as a pro-drug of a thymidylate synthase inhibitor, because it is metabolized into 5fluorouridine [START_REF] Boothman | Exploitation of elevated pyrimidine deaminating enzymes for selective chemotherapy[END_REF]. Zebularine is also a stable compound with a weaker inhibition activity and cytotoxicity than azacytosine analogs [START_REF] Flotho | The DNA methyltransferase inhibitors azacitidine, decitabine and zebularine exert differential effects on cancer gene expression in acute myeloid leukemia cells[END_REF]. Its mode of action is different from 5aza and 5azadC, since it forms a very stable, but reversible complex with DNMTs that showed a slow dissociation kinetic [START_REF] Champion | Mechanistic Insights on the Inhibition of C5 DNA Methyltransferases by Zebularine[END_REF][START_REF] Van Bemmel | DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site[END_REF].

Second, a pro-drug approach was chosen aiming at releasing the active molecule in the organism. This approach resulted in the generation of NPEOC-DAC [START_REF] Byun | 2'-Deoxy-N4-[2-(4nitrophenyl)ethoxycarbonyl]-5-azacytidine: a novel inhibitor of DNA methyltransferase that requires activation by human carboxylesterase 1[END_REF], CP-4200 [START_REF] Brueckner | Delivery of 5azacytidine to human cancer cells by elaidic acid esterification increases therapeutic drug efficacy[END_REF] and SGI-110 [START_REF] Chuang | S110, a 5-Aza-2′-Deoxycytidine-Containing Dinucleotide, Is an Effective DNA Methylation Inhibitor in vivo and Can Reduce Tumor Growth[END_REF][START_REF] Yoo | Delivery of 5-aza-2'deoxycytidine to cells using oligodeoxynucleotides[END_REF]. SGI-110 or guadecitabine, composed of a 5azad-CpG dinucleotide, is a pro-drug of 5azadC, it entered clinical trials and is the most promising pro-drug with a comparable in vitro and in vivo DNMT inhibition activity. The advantage of SGI-110 is that it is less sensitive to cytidine deaminase than 5azadC, which improves its metabolic stability.

Third, non-nucleoside compounds were investigated. However, most of them lack specificity, show weak activity against the enzyme or do not induce a strong DNA methylation inhibition in cellular models. Several natural products were reported as DNMTi (Figure 17.2), for example, (-)epigallocatechin-3-gallate (EGCG) [START_REF] Fang | Tea Polyphenol (-)-Epigallocatechin-3-Gallate Inhibits DNA Methyltransferase and Reactivates Methylation-Silenced Genes in Cancer Cell Lines[END_REF] and laccaic acid [START_REF] Fagan | Laccaic Acid A Is a Direct, DNAcompetitive Inhibitor of DNA Methyltransferase 1[END_REF]. These natural products lack specificity and their mode of action was afterwards questioned, as the one of genistein [START_REF] Fang | Reversal of Hypermethylation and Reactivation of p16INK4a, RARβ, and MGMT Genes by Genistein and Other Isoflavones from Soy[END_REF], psammaplin A [START_REF] Baud | Defining the mechanism of action and enzymatic selectivity of psammaplin A against its epigenetic targets[END_REF][START_REF] Pereira | Indole-Derived Psammaplin A Analogues as Epigenetic Modulators with Multiple Inhibitory Activities[END_REF] and curcumin [START_REF] Liu | Curcumin is a potent DNA hypomethylation agent[END_REF]. Indeed, indirect or off-target effects seem to be responsible for the observed inhibition of DNA methylation. All these compounds are known as multi-target compounds [START_REF] Lopez | Drug Discovery Methods[END_REF], Pechalrieu, 2017). Similarly, Hann's team highlighted an indirect decrease in DNMT1 expression in cancer cells (lung cancer and hepatocarcinoma) via ERK1/2-and AMPKa-mediated inhibition of the transcription factor Sp1 after treatment with two natural products, β-elemene (Zhao, 2015) and ursolic acid [START_REF] Yie | Ursolic acid inhibited growth of hepatocellular carcinoma HepG2 cells through AMPKalpha-mediated reduction of DNA methyltransferase 1[END_REF].

Nanaomycin A is a more specific compound, because it was found to target specifically DNMT3B, and shown to induce genomic DNA methylation inhibition in colon cancer cell line (Kuck, 2010a).

Nevertheless, in a recent study, Penter et al. did not observe any DNA methylation inhibition by nanaomycin A in four neuroblastoma cell lines, although they observed an interesting synergistic activity when used in combination with doxorubicin [START_REF] Penter | A rapid screening system evaluates novel inhibitors of DNA methylation and suggests F-box proteins as potential therapeutic targets for high-risk neuroblastoma[END_REF]. To conclude, none of the natural products described to date clearly targets the DNMTs directly in cells and their mechanism of action is controversial (Li, 2010[START_REF] Medina-Franco | Natural products as DNA methyltransferase inhibitors: a computer-aided discovery approach[END_REF][START_REF] Suh | Strawberry Fields Forever?[END_REF].

The same difficulty to identify potent and specific non-nucleoside DNMTi is found among the synthetic compounds. For example, drug repurposing showed that hydralazine and procainamide are week DNMTi [START_REF] Candelaria | DNA Methylation-Independent Reversion of Gemcitabine Resistance by Hydralazine in Cervical Cancer Cells[END_REF][START_REF] Chuang | Comparison of biological effects of non-nucleoside DNA methylation inhibitors versus 5-aza-2′-deoxycytidine[END_REF]. These two FDA approved drugs are vasodilator and anti-arrhythmic compounds, respectively, that were described as DNMTi because patients developed auto-immune disease (lupus erythematosus) correlated to an abnormal DNA methylation pattern of Tcells [START_REF] Cornacchia | Hydralazine and procainamide inhibit T cell DNA methylation and induce autoreactivity[END_REF]. Despite the weak DNMT inhibition activity of hydralazine, a molecular modeling study (Singh, 2009a) showed that hydralazine could be involved in a complex network of hydrogen bonds into the cytidine pocket. Today, hydralazine is still extensively studied, especially in combination with valproic acid (VPA), an HDAC inhibitor (Dueñas-Gonzalez, 2014). Procainamide was also described to potentially interact within the catalytic side by molecular modeling (Singh, 2009a), while biophysical studies previously demonstrated that it acted as DNA ligand and decreased the processivity of DNMTs (Lee, 2005). Procainamide and procaine were indeed described as DNA ligands with a certain specificity for CG-rich regions, potentially targeting the DNA binding of the DNMT as described above in paragraph 17.1 [START_REF] Villar-Garea | Procaine Is a DNA-demethylating Agent with Growth-inhibitory Effects in Human Cancer Cells[END_REF]. Procainamide was an interesting starting point for drug design and several constrained derivatives were synthesized and tested [START_REF] Castellano | Constrained analogues of procaine as novel small molecule inhibitors of DNA methyltransferase-1[END_REF][START_REF] Castellano | Synthesis and Biochemical Evaluation of 2-Isoxazoline Derivatives as DNA Methyltransferase 1 Inhibitors[END_REF]. In parallel, procainamide conjugated to RG108, an in silico identified pharmacophore (see paragraph below), induced a synergy in the inhibition of the enzymatic activity of DNMT3A and DNMT1 [START_REF] Halby | Rapid Synthesis of New DNMT Inhibitors Derivatives of Procainamide[END_REF]. Initially, it was hypothesized that the procainamide moiety of these compounds would bind to DNA and RG108 moiety would bind to DNMT catalytic pocket. Later, a molecular modeling study suggested that the conjugates could occupy both SAM and cytidine pockets [START_REF] Yoo | Molecular Modeling Studies of the Novel Inhibitors of DNA Methyltransferases SGI-1027 and CBC12: Implications for the Mechanism of Inhibition of DNMTs[END_REF] explaining the increased activity (cf. paragraph 17.1).

In parallel, virtual screening-based studies led to the identification of RG108 [START_REF] Brueckner | Epigenetic Reactivation of Tumor Suppressor Genes by a Novel Small-Molecule Inhibitor of Human DNA Methyltransferases[END_REF][START_REF] Siedlecki | Discovery of Two Novel, Small-Molecule Inhibitors of DNA Methylation[END_REF][START_REF] Stresemann | Functional Diversity of DNA Methyltransferase Inhibitors in Human Cancer Cell Lines[END_REF], DC_517 (Chen, 2014), thienoquinazolinone-adenine 33h [START_REF] Newton | Structure-Guided Identification of DNMT3B Inhibitors[END_REF] and pyrazolone/pyridazine derivatives [START_REF] Huang | A novel class of selective non-nucleoside inhibitors of human DNA methyltransferase 3A[END_REF]. Recent experimental studies revealed that RG108 has only a weak activity against human DNMTs [START_REF] Halby | Rapid Synthesis of New DNMT Inhibitors Derivatives of Procainamide[END_REF][START_REF] Suzuki | Design, synthesis, inhibitory activity, and binding mode study of novel DNA methyltransferase 1 inhibitors[END_REF] and more potent derivatives were since synthesized such as maleimide [START_REF] Suzuki | Design, synthesis, inhibitory activity, and binding mode study of novel DNA methyltransferase 1 inhibitors[END_REF], constraint analogs [START_REF] Asgatay | Synthesis and evaluation of analogues of N-phthaloyl-L-tryptophan (RG108) as inhibitors of DNA methyltransferase 1[END_REF] and other derivatives [START_REF] Rondelet | Inhibition studies of DNA methyltransferases by maleimide derivatives of RG108 as non-nucleoside inhibitors[END_REF][START_REF] Rotili | Properly substituted analogues of BIX-01294 lose inhibition of G9a histone methyltransferase and gain selective anti-DNA methyltransferase 3A activity[END_REF]. Nevertheless, their ability to inhibit genomic DNA methylation has still to be established for all these inhibitors. New generation compounds seem to be more promising, as they showed DNA methylation inhibition and/or gene reexpression in cells. DC_517 and Cpd 33h showed micromolar inhibition activity against DNMT1 (IC50 = 1.7 µM) (Chen, 2014) and DNMT3B (IC50 = 8 µM) [START_REF] Newton | Structure-Guided Identification of DNMT3B Inhibitors[END_REF], respectively. DC_517 reduced proliferation and induced apoptosis in HCT116 colorectal cancer cell line (Chen, 2014).

Regarding pyrazolone/pyridazine derivatives, enzymology studies interestingly suggested that they act as allosteric inhibitors with high-micromolar range activity on purified DNMTs [START_REF] Huang | A novel class of selective non-nucleoside inhibitors of human DNA methyltransferase 3A[END_REF] but their efficacy in cells needs to be confirmed. Virtual screening followed by structure optimization also led to the discovery of GSK3484862 [START_REF] Pappalardi | Discovery of a firstin-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia[END_REF], a highly potent DNMT1-selective inhibitor with IC50 = 0.23 µM vs IC50 > 50 µM for DNMT3A/B. In cells, GSK3484862 decreased promoter methylation and reinduceds expression of VIM gene, and it also induced global decrease of DNA methylation levels in murine embryonic stem cells with limited toxicity [START_REF] Azevedo Portilho | The DNMT1 inhibitor GSK-3484862 mediates global demethylation in murine embryonic stem cells[END_REF].

Several High Throughput Screening (HTS) campaigns were performed and allowed to identify new families of DNMTi. 3-Chloro-3-nitroflavanones were identified [START_REF] Ceccaldi | C5-DNA Methyltransferase Inhibitors: From Screening to Effects on Zebrafish Embryo Development[END_REF] with submicromolar activity against Dnmt3a/3L complex and exhibiting a phenotype in zebrafish embryos similar to the one observed with the 5aza. This scaffold was further optimized and 3-bromo-3nitroflavanones showed improved stability and cellular activity in several cancer models in vitro and ex vivo [START_REF] Lobo | Targeting Germ Cell Tumors with the Newly Synthesized Flavanone-Derived Compound MLo1302 Efficiently Reduces Tumor Cell Viability and Induces Apoptosis and Cell Cycle Arrest[END_REF][START_REF] Marques-Magalhães | Anti-neoplastic and demethylating activity of a newly synthetized flavanone-derived compound in Renal Cell Carcinoma cell lines[END_REF][START_REF] Pechalrieu | Synthesis of novel 3-halo-3-nitroflavanones and their activities as DNA methyltransferase inhibitors in cancer cells[END_REF]. A naphtoquinone, diclone, and other flavonoids were identified in another screening campaigns, opening the path to study the impact on the epigenome of plants, animals and humans when addressing the toxicology of pesticides [START_REF] Ceccaldi | Identification of Novel Inhibitors of DNA Methylation by Screening of a Chemical Library[END_REF]. Molecular modeling studies suggested that the most potent compounds occupy both the cytosine and SAM pockets [START_REF] Ceccaldi | C5-DNA Methyltransferase Inhibitors: From Screening to Effects on Zebrafish Embryo Development[END_REF]. SW155246, an aromatic sulfonamide, was also identified by HTS [START_REF] Kilgore | Identification of DNMT1 selective antagonists using a novel scintillation proximity assay[END_REF]. Interestingly, this compound was able to induce a weak methylation inhibition and reactivation of tumor suppressor genes (TSGs) in human lung carcinoma.

Acridine derivatives, known to intercalate into DNA, were described to modulate DNA methylation [START_REF] Hossain | DNA-intercalators causing rapid reexpression of methylated and silenced genes in cancer cells[END_REF]. Among them, derivative 517328 was the most potent analog inhibiting DNMT1 in vitro (30% inhibition at 10 µM) and DNA methylation of TSGs in colon and pancreatic cancer cell lines. This compound demonstrated that DNA binders inhibit DNMTs, however the specificity of such inhibitors remains an important limitation to their use. SGI-1027 is another weak DNA ligand that was identified to be a DNMT1 inhibitor [START_REF] Datta | A New Class of Quinoline-Based DNA Hypomethylating Agents Reactivates Tumor Suppressor Genes by Blocking DNA Methyltransferase 1 Activity and Inducing Its Degradation[END_REF]. Molecular modeling studies suggested that SGI-1027 could inhibit DNMTs by occupying both cytidine and SAM pockets [START_REF] Yoo | Molecular Modeling Studies of the Novel Inhibitors of DNA Methyltransferases SGI-1027 and CBC12: Implications for the Mechanism of Inhibition of DNMTs[END_REF] and Gros et al. [START_REF] Gros | New Insights on the Mechanism of Quinoline-based DNA Methyltransferase Inhibitors[END_REF] confirmed, by biophysical studies, that it interacts with DNA and functions as a SAM non-competitive but DNA-competitive inhibitor. SGI-1027 was described to induce DNMT1 degradation and TSG re-expression in colon cancer cell lines and was quickly considered as the most promising starting point for DNMTi design. Several derivatives [START_REF] Gamage | Structure-activity relationships for 4-anilinoquinoline derivatives as inhibitors of the DNA methyltransferase enzyme DNMT1[END_REF][START_REF] Rilova | Design, Synthesis and Biological Evaluation of 4-Amino-N-(4-aminophenyl)benzamide Analogues of Quinoline-Based SGI-1027 as Inhibitors of DNA Methylation[END_REF][START_REF] Valente | Selective non-nucleoside inhibitors of human DNA methyltransferases active in cancer including in cancer stem cells[END_REF] succeeded in increasing significantly DNMT inhibition potency with the meta/meta analog, but no potent inhibition of genomic DNA methylation or TSG re-expression has so far been shown.

Further investigations to characterize the mode of action of this compound family indicated that the meta/meta analog strongly interacted with DNA and inhibited DNMT by DNA interaction and destabilization of the DNMT/DNA/SAM complex [START_REF] Gros | New Insights on the Mechanism of Quinoline-based DNA Methyltransferase Inhibitors[END_REF].

Finally, Halby et al. carried out a rational design based on bisubstrate analogues and identified quinoline-quinazoline DNMTi with compound 68 as their lead compound. This DNMTi strongly inhibits DNMT3A (EC50 = 1.1 µM) with a good selectivity over DNMT1 (EC50 = 100 µM). Besides it potently reduced promoter methylation of CDKN2A together with an increase of its expression level [START_REF] Halby | Rational design of bisubstratetype analogs as inhibitors of DNA methyltransferases in cancer cells[END_REF].

To conclude, to date only nucleoside DNMTi were FDA-approved and SGI-110 is a promising prodrug of 5azadC, evaluated in clinical trials both in hematological and solid cancers. An increasing number of new non-nucleoside DNMTi was published in the last few years, but most lack selectivity or demonstrated impact on genomic DNA methylation and TSG re-expression, except for GSK3484862, an allosteric inhibitor, that is to date the most promising chemical probe for DNMT.

Several strategies are therefore implemented to discover more potent DNMTi.

Potential applications of DNMT inhibitors

DNA methylation is crucial for the control of gene expression and cell proliferation. Indeed, DNA methylation ion the CpG islands of gene promoters leads to the silencing of the corresponding gene.

This participates in the dynamics of the gene expression regulation in cells and is crucial for normal cell functions. However, many factors, including diet, stress, environmental conditions, etc. can lead to abnormal DNA methylation patterns. These deregulations are often responsible for dysfunctions and development of diseases such as cancers, neurological and cardiovascular diseases, and abnormal plant growth (Figure 17.3). As all epigenetic modifications, DNA methylation is reversible and therefore can be chemically reversed. The main application of DNMTi is by far their use as anticancer agents.

However, the importance of DNA methylation in various biological contexts has led to increasing research in the context of other pathologies, such as neurodegenerative and cardiovascular diseases and in other fields, such as plant growth optimization.

DNMTi application in cancers

In cancer, hypermethylation of TSG promoter regions is observed together with a global hypomethylation [START_REF] Esteller | Epigenetics in Cancer[END_REF]. The hypermethylation of promoters of genes, such as P53, P16, P15, RAR2, HIC1 and RASSF1A, results in their silencing and participates in tumor formation, maintenance and proliferation.

a. Nucleoside analogues

As single agent

As described above, 5aza and 5azadC are the most extensively used DNMTi. These nucleoside analogues are able to decrease TSG-promoter methylation levels in cancer cells and induce TSG reexpression, resulting in cell reprogramming and, eventually, cell cycle arrest and apoptosis [START_REF] Fahy | DNA methyltransferase inhibitors in cancer: a chemical and therapeutic patent overview and selected clinical studies[END_REF]. A historical overview of their development and application is described in [START_REF] Issa | Targeting DNA Methylation[END_REF] As described above, to overcome 5aza and 5azadC drawbacks (i.e. chemical instability, poor delivery and side-effects), other nucleoside analogs were developed (Figure 17.2), but they need to be used at higher doses due to their lower efficacy and up to date no clinical trial were undertaken with these compounds.

Despite their promise for non-solid tumor treatment, the direct use of nucleoside analogs as a single therapeutic agent is quite limited considering their instability, their side effects and their lower therapeutic index in solid tumors.

Pro-drugs of nucleoside analogues

Pro-drugs of 5aza and 5azadC, which delay the release of the active molecule in the organism and lower its degradation, showed a real improvement of the drugs. CP-4200 is a lipophilic ester of 5aza patented for its better cellular uptake and activity in vivo in an orthotropic acute lymphoblastic leukemia (ALL) mouse model [START_REF] Silverman | Analogues d'azacytidine et leurs utilisations[END_REF], but no clinical trial was undertaken up to date. SGI-110 (guadecitabine) is a 5azadC pro-drug, which avoids its decomposition by deaminase [START_REF] Chuang | S110, a 5-Aza-2′-Deoxycytidine-Containing Dinucleotide, Is an Effective DNA Methylation Inhibitor in vivo and Can Reduce Tumor Growth[END_REF]. In the last few years many clinical trials were started with SGI-110 alone (for example in phase III clinical trials for AML (Acute Myeloid Leukemia), MDS (Myelodysplastic Syndrome) and CMML (Chronic Muelomonocytic Leukemia), in phase II for gastrointestinal cancer and in phase I for testicular cancer) and in combination with immunotherapies (for example in phase II for kidney, ovarian, melanoma and (non-) small cell lung cancers).

Nucleoside DNMTi in combination with other drugs

With other epidrugs

Nucleoside DNMTi were studied in combinations with other epidrugs, as for example 5azadC was combined with trichostatin A (TSA), an HDACi, in colorectal carcinoma cell line. Whereas TSA alone was not able to cause re-expression of MLH1, TIMP3, P15 and P16 genes, a pre-treatment with a low dose of 5azadC led to their re-expression [START_REF] Cameron | Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer[END_REF]. The combination of 5azadC and valproic acid (VPA), another HDACi, was evaluated in AML and MDS (Yang, 2005) and tested in phase II clinical trials (NCT00414310). However, no improvement in outcome was reported. The association of 5aza or 5azadC with the FDA-approved HDACi vorinostat (SAHA) was proven to be of interest in hematological cancers [START_REF] Silverman | A phase I trial of epigenetic modulators vorinostat, in combination with azacitidine (azaC) in patients with the myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML): a study of the New York Cancer Consortium[END_REF], in colorectal cancer cells [START_REF] Najem | Synergistic Effect of Epigenetic Inhibitors Decitabine and Suberoylanilide Hydroxamic Acid on Colorectal Cancer In vitro[END_REF] and in vivo in a colon carcinoma CT26 experimental lung metastasis mouse model [START_REF] Yang | Decitabine and Vorinostat Cooperate To Sensitize Colon Carcinoma Cells to Fas Ligand-Induced Apoptosis In Vitro and Tumor Suppression In Vivo[END_REF]. In addition, 5aza was reported to be active in combination with entinostat and mocetinostat, benzamide inhibitors of HDACi [START_REF] Fandy | Early epigenetic changes and DNA damage do not predict clinical response in an overlapping schedule of 5-azacytidine and entinostat in patients with myeloid malignancies[END_REF] and these combinations are tested in phase I and II clinical trials in hematological cancers. 5aza/entinostat combination is also studied in solid cancers, such as advanced non-small cell lung cancer (NCT01886573) [START_REF] Juergens | Combination Epigenetic Therapy Has Efficacy in Patients with Refractory Advanced Non-Small Cell Lung Cancer[END_REF], advanced breast cancer (NCT01349959) and metastatic colorectal cancer (NCT01105377) (see Table 17.1 for some examples). Combination of 5aza/pracinostat, an hydroxamic acid based HDACi, is in phase III for the treatment of AML (NCT03151408).

With "classical" chemo-and immunotherapies

Great promises arise from the use of nucleoside DNMTi in combination with chemotherapies or immunotherapies in tumors (Table 17.1). The rational of these combination approaches relies on the fact that, due to their capacity to globally affect cells and restore cell functions, DNMTi can increase the sensitivity to other anticancer agents, resulting in higher efficacy [START_REF] Ahuja | Harnessing the potential of epigenetic therapy to target solid tumors[END_REF][START_REF] Ahuja | Epigenetic Therapeutics: A New Weapon in the War Against Cancer[END_REF][START_REF] Azad | The future of epigenetic therapy in solid tumourslessons from the past[END_REF][START_REF] Cameron | Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer[END_REF]. A plethora of clinical trials, involving multi-anti-cancer agents in combination In parallel, several studies described an enhancement of radiosensitivity by 5azadC treatment in lung cancer A549 and glioblastoma U373MG cells [START_REF] Kim | DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity[END_REF], SaOS2, HOS, and U2OS osteosarcoma cells (Li, 2014) and MDA-MB-231 and MDA-MB-435 breast cancer cell lines (Wang, 2013a). In all cases, the increase in radiosensitivity was correlated with cell cycle-, apoptosis-and radiosensitivityrelated gene up-regulation and G2/M cell arrest.

In summary, nucleoside DNMTi are frequently used for hematological cancers. New clinical trials are ongoing to optimize the treatment schedule, using a low-dose strategy, and evaluate DNMTi efficacy in solid tumors. SGI-110 could eventually overcome the compound stability limitation. In parallel, their use in combination with other anticancer agents is giving promising results increasing the efficacy of the treatments and decreasing the side effects, since lower doses of each drug are used.

b. Non-nucleoside DNMTi

As described above, several non-nucleoside DNMTi have been reported, but none obtained the same potency and validation as 5aza and 5azadC. Still ongoing studies and clinical trials are required to better understand the therapeutic potential of DNA methylation [START_REF] Erdmann | Targeting DNA methylation with small molecules: what's next?[END_REF]. Hydralazine (de la [START_REF] De La | DNA Demethylating Activity of Hydralazine in Cancer Cell Lines[END_REF], Graça, 2014b) failed as single therapeutic agent in phase II for breast and rectal cancer [START_REF] Wang | Association between CpG Island Methylation of the WWOX Gene and Its Expression in Breast Cancers[END_REF]. Its combination with VPA (Duenas-Gonzalez, 2008) was completed in phase II against cervical cancer (NCT00404326) and refractory solid tumors (NCT00404508) more than 10 years ago and no further studies had been started since.

Indirect effects on DNA methylation are also of therapeutic interest. In the presence of disulfiram, in vitro DNMT1 activity was shown to be reduced, and global hypomethylation was observed in prostate cancer cell lines, with decrease in APC and RAR-β promoter methylation accompanied by their reexpression. Xenograft tumor volumes were shown to be reduced under disulfiram treatment independently of the dose (from 10 to 40 mg.kg -1 ) [START_REF] Lin | Disulfiram is a DNA demethylating agent and inhibits prostate cancer cell growth[END_REF]. However, a completed Disulfiram phase II study prostate cancer showed a limited impact (NCT01118741). Combination of disulfiram with copper improved its potency in refractory cancers (Li, 2020a). This combination is currently in phase II against metastatic breast (NCT03323346) and pancreatic (NCT03714555) cancers and in phase III against glioblastoma (NCT02678975).

RG108, found by virtual screening, was shown to inhibit methylation in NALM6 (leukemia cell line)

and in HCT116 (colorectal cancer cell line), inducing re-expression of P16 and TIMP3 [START_REF] Stresemann | Functional Diversity of DNA Methyltransferase Inhibitors in Human Cancer Cell Lines[END_REF]. In prostate cancer cell lines (LNCaP and 22Rv1), it decreased DNMT activity together with cell growth inhibition and apoptosis (Graça, 2014a). However, the mechanism by which RG108 treatment resulted in a decrease of the methylation level in cells remains to be understood.

Nevertheless, since it is commercially available, it was used as a tool to inhibit DNA methylation in several cellular models (see below).

DNMTi application in neurological and psychiatric disorders

DNA methylation, together with other epigenetic modifications, is also deregulated in neurological diseases and psychiatric disorders, such as schizophrenia, epilepsy or bipolar disorder (for review, [START_REF] Murshid | Epigenetic Changes and Its Intervention in Age-Related Neurodegenerative Diseases[END_REF]). However, to date, no DNMTi has been approved for these diseases, the main hurdle being the poor capability of the existing DNMTi to cross the blood-brain barrier (BBB).

Nevertheless, interesting results, listed here below, highlight the great interest of targeting DNA methylation in neurological and psychiatric disorders.

a. Memory formation

Memory loss is involved in many neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, schizophrenia or post-traumatic stress disorders. DNA methylation is implicated in memory formation, as it regulates gene transcription in central nervous system (CNS) and is required in longterm memory formation [START_REF] Day | DNA methylation and memory formation[END_REF]. Additionally, DNA methylation was demonstrated to be involved in synaptic plasticity as Dnmt1/Dnmt3a double knockout in mice exhibit long-term potentiation (LTP) deficiencies (Feng, 2010). To better understand the implication of DNA methylation in various brain regions, rats were treated with RG108 by using the object-in-place paradigm, a test based on the memory of the positioning of objects in an enclosed space, which requires both hippocampus and perirhinal cortex brain regions. Using this model, Michnick et al.

demonstrated that DNA methylation was required for long-term but not short-term memory [START_REF] Mitchnick | Differential contributions of de novo and maintenance DNA methyltransferases to object memory processing in the rat hippocampus and perirhinal cortexa double dissociation[END_REF]. In parallel, expression of Pp1, a memory suppressor gene, and Reln, coding for reelin, a positive memory regulator, was shown to be controlled by promoter methylation. Following fear conditioning, an increase in Pp1 promoter methylation and a decrease in Reln promoter methylation levels was observed in adult rat hippocampus. Infusion of 5aza in adult mouse brain inverted these effects, decreased Pp1 methylation and enhanced a the low methylation level of Reln, which inversely correlated with the expression of both genes [START_REF] Miller | Covalent Modification of DNA Regulates Memory Formation[END_REF]. In rats trained for contextual fear memory, the authors observed that intra-anterior cingulate cortex infusions of 5aza or zebularine, 30 days post-training, disrupted remote memory [START_REF] Miller | Cortical DNA methylation maintains remote memory[END_REF]. Reelin is also implicated in synaptic plasticity that is involved in storage memory. Levenson et al. demonstrated that zebularine induced a significant methylation decrease in one of the two CpG islands of the Reln promoter, whereas the other CpG methylation level remained steady [START_REF] Levenson | Evidence That DNA (Cytosine-5) Methyltransferase Regulates Synaptic Plasticity in the Hippocampus[END_REF]. The impact of DNMTi in synaptic plasticity was studied by Nelson et al. in hippocampus slices, where 5azadC was reported to decrease the genomic DNA methylation level, concomitantly with a reduction of miniature excitatory post-synaptic current frequencies in neurons, impacting neuronal activity [START_REF] Nelson | Activity-Dependent Suppression of Miniature Neurotransmission through the Regulation of DNA Methylation[END_REF]. Tetrodotoxin, known to decrease neuronal activity, also induced a loss of methylation and the same effects were observed both by using RG108 or by knocking down Dnmt1 and Dnmt3a [START_REF] Meadows | DNA methylation regulates neuronal glutamatergic synaptic scaling[END_REF].

Treatment of honeybees with RG108 also induced relearning and bees were able to discriminate between a known and a new odor [START_REF] Biergans | DNA Methylation Adjusts the Specificity of Memories Depending on the Learning Context and Promotes Relearning in Honeybees[END_REF].

Altogether, these studies show that DNA methylation is implicated in memory regulation, long-term memory and synaptic plasticity. DNMTi were shown to interfere with the long-term memory and to enhance synapse receptiveness in neurons (synaptic upscaling). Consequently, DNMTi were shown to impair memory, suggesting a positive effect on post-traumatic stress disorders (PTSD) caused by longlasting traumatic memory (see below). Animal studies using 5azadC were, in most cases, carried out by direct injection in brain regions or on slice cultures. However, 5azadC injected intraperitonially in mice led also to an increase of Bdnf expression and a lower global DNA methylation in the hippocampus [START_REF] Sales | Antidepressant-like effect induced by systemic and intra-hippocampal administration of DNA methylation inhibitors[END_REF]. These effects on memory functions were hypothesized as limiting factor for the use of decitabine in MDS patients [START_REF] Aydin | Could decitabine treatment impair memory functions in humans?[END_REF].

b. Schizophrenia

Schizophrenia is a cognition disorder often characterized by hallucination, paranoia and failure to adopt a "normal" behavior in social situations. It is now quite clearly established that a deregulation of the GABA ((γ-aminobutyric acid)ergenic/glutamatergic) network in the hippocampus and cortex is characteristic of psychotic disorders including schizophrenia [START_REF] Lewis | Cortical inhibitory neurons and schizophrenia[END_REF]. More specifically, a down-regulation of GABAergic genes, such as glutamic acid decarboxylase67 (GAD67) and RELN, was measured in post-mortem samples of schizophrenia patients [START_REF] Guidotti | Decrease in Reelin and Glutamic Acid Decarboxylase67 (GAD67) Expression in Schizophrenia and Bipolar Disorder: A Postmortem Brain Study[END_REF]. This down-regulation was correlated with an hypermethylation of their CpG island promoter regions [START_REF] Chen | On the epigenetic regulation of the human reelin promoter[END_REF][START_REF] Grayson | Reelin promoter hypermethylation in schizophrenia[END_REF] and with an increase in DNMT1 [START_REF] Veldic | DNA-methyltransferase 1 mRNA is selectively overexpressed in telencephalic GABAergic interneurons of schizophrenia brains[END_REF]) and DNMT3A [START_REF] Zhubi | An upregulation of DNAmethyltransferase 1 and 3a expressed in telencephalic GABAergic neurons of schizophrenia patients is also detected in peripheral blood lymphocytes[END_REF] mRNA expression in schizophrenia post-mortem cortical GABAergic neurons. In a model of prenatal-stress mice exhibiting a schizophrenia-like behavior, high levels of Dnmt1 and Dnmt3a expression occurred in GABAergic neurons [START_REF] Matrisciano | Epigenetic modifications of GABAergic interneurons are associated with the schizophrenia-like phenotype induced by prenatal stress in mice[END_REF]. Additionally, a knock-down of Dnmt1 in mouse primary cortical cultures showed that Reln expression is controlled by Dnmt1 [START_REF] Noh | DNA methyltransferase 1 regulates reelin mRNA expression in mouse primary cortical cultures[END_REF].

In this context, DNMTi were tested to decrease the methylation level and restore a normal expression of GAD67 and RELN. 5aza, zebularine and procainamide were tested in cell cultures and an increase in the expression of GAD67 and RELN and a decrease in DNMT1 expression were observed [START_REF] Kundakovic | DNA Methyltransferase Inhibitors Coordinately Induce Expression of the Human Reelin and Glutamic Acid Decarboxylase 67 Genes[END_REF]. However, 5aza and zebularine, the most active molecules in the cellular context, present limiting potential to cross the blood-brain barrier (BBB).

c. Bipolar Disorders

Bipolar disorders are maniac-depressive disorders characterized by a succession of elevated mood and depression periods. Aberrant DNA methylation patterns are implicated in the etiology of bipolar disorders and, very similarly to schizophrenia, patients with bipolar disorders show a down-regulation of GAD67 and RELN [START_REF] Guidotti | Decrease in Reelin and Glutamic Acid Decarboxylase67 (GAD67) Expression in Schizophrenia and Bipolar Disorder: A Postmortem Brain Study[END_REF]. Because of the lack of relevant animal models to study bipolar disorders, very few studies involving DNMTi have been carried out. Mainly, VPA, an HDACi, was showed to decrease DNA methylation without decrease in DNMT expression [START_REF] Aizawa | Valproate administration to mice increases hippocampal p21 expression by altering genomic DNA methylation[END_REF]. VPA is FDA approved for bipolar disorders and in clinical studies in combination with antipsychotics.

d. Epilepsy

Epilepsy is a group of neurological diseases for whichcauses are unknown in most cases. Epileptic patients are subject to recurrent unprovoked seizures, which are brief to quite long periods of intense shaking.

In temporal lobe epilepsy, the most common epilepsy syndrome in human, low levels of reelin were observed in brain specimens [START_REF] Heinrich | Reelin Deficiency and Displacement of Mature Neurons, But Not Neurogenesis, Underlie the Formation of Granule Cell Dispersion in the Epileptic Hippocampus[END_REF] in correlation with a hypermethylation of its promoter region [START_REF] Kobow | Increased reelin promoter methylation is associated with granule cell dispersion in human temporal lobe epilepsy[END_REF]. A genome-wide study revealed a global DNA hypermethylation in chronic epileptic rat correlated to a decrease in reelin gene expression at the mRNA level [START_REF] Kobow | Deep sequencing reveals increased DNA methylation in chronic rat epilepsy[END_REF].

Moreover, Gria2, a Glu2A subunit of ionotropic glutamate receptor, has been identified to play a role in epilepsy, and its hypermethylation and corresponding decrease in mRNA expression was correlated to the intensity and frequency of seizure in rats [START_REF] Machnes | DNA Methylation Mediates Persistent Epileptiform Activity In Vitro and In Vivo[END_REF]. In an equivalent rat model of kainic acid-induced epilepsy, bisulfite sequencing showed a hypermethylation of Grin2b/Nr2b, resulting in a lower level of the GRIN2B protein, another glutamate receptor subunit, together with a hypomethylation of the Bdnf gene in epileptic hippocampus [START_REF] Parrish | Status Epilepticus Triggers Early and Late Alterations in Brain-Derived Neurotrophic Factor and NMDA Glutamate Receptor GRIN2B DNA Methylation Levels in the Hippocampus[END_REF]. RG108 treatment on kainite-treated hippocampal slice cultures showed an inhibition of the Gria2 hypermethylation and a beneficial increase in Gria2 activity [START_REF] Machnes | DNA Methylation Mediates Persistent Epileptiform Activity In Vitro and In Vivo[END_REF]. Zebularine also decreased the methylation level of Grin2b/Nr2b, concomitantly with an increase in GRIN2B protein level [START_REF] Parrish | Status Epilepticus Triggers Early and Late Alterations in Brain-Derived Neurotrophic Factor and NMDA Glutamate Receptor GRIN2B DNA Methylation Levels in the Hippocampus[END_REF]. All these findings are in favor of applying DNMTi to epilepsy treatment. It was also demonstrated that treatment with RG108 led to a lower methylation level of RASgrf1gene and increase its expression, which was associated with the suppression of epileptic seizure in epileptic mice model (Chen, 2017).

e. Post-traumatic stress disorder

Post-traumatic stress disorders (PTSD) appear in patients who experienced a psychologically traumatic event, such as violent death witnessing, domestic violence, child abuse, etc. and result in severe anxiety associated with memory avoidance, flashbacks, nightmares and emotional arousal. For example, war veterans commonly suffer from PTSD.

In PTSD patients, altered DNA methylation was observed in the peripheral blood immune cells.

Together with other genes [START_REF] Uddin | Epigenetic and immune function profiles associated with posttraumatic stress disorder[END_REF], BDNF was reported to be hypermethylated [START_REF] Smith | Differential immune system DNA methylation and cytokine regulation in post-traumatic stress disorder[END_REF]. In rat models, epigenetic modifications were identified to play a role in fear memory and Miller et al.

demonstrated, that Dnmt3a and 3b are up-regulated, whereas level of Dnmt1 remains steady in rat hippocampus, in response to contextual fear conditioning [START_REF] Miller | Covalent Modification of DNA Regulates Memory Formation[END_REF]. Models of maltreated rats compared to normal animals also showed an increase in DNA methylation levels of Bdnf, associated with a decrease of Bdnf expression. This modification is a lasting effect, which was observed in adulthood and passed to the next generation [START_REF] Roth | Lasting Epigenetic Influence of Early-Life Adversity on the BDNF Gene[END_REF]. DNA methylation of BDNF contributes to learned fear and BDNF is important for the persistence of the pathological fear. Therefore, DNMTi could be envisaged to erase the memory of the trauma in PTSD patients [START_REF] Zovkic | LPS: lipopolysaccharide LTP: long-term potentiation MDS: myelodiysplasic syndrome MeCP2: methyl-CpG-binding protein 2 mPFC: medial prefrontal cortex MSC: mesenchymal stromal cells NHL: non-Hodgkin's lymphomas NMS: neonatal maternal separation NSC: neuronal stem cell PARP: poly-(ADP-ribose) polymerase PARPi: PARP inhibitor PD: Parkinson's disease PTSD: post-traumatic stress disorder SAHA: suberoylanilide hydroxamic acid, vorinostat (Zolinza®) SAM: S-adenosyl-L-methionine SAH: S-adenosyl-L-homocysteine SAR: seasonal allergic rhinitis SFRP4: secreted frizzled-related protein SHS: second-hand smoke Th: T-helper TSA: trichostatin A TSG: tumor suppressor gene VPA: valproic acid XRCC1: X-ray repair cross-complementing protein[END_REF].

Administration of DNMTi (5aza or zebularine) induces Bdnf expression even in adult animals. In PTSD, an over-consolidation of the fear memory is observed and nucleoside DNMTi and RG108 were able to block the contextual fear memory formation. RG108 was also tested directly in rat brains and shown to abolish long-term fear memory [START_REF] Miller | Cortical DNA methylation maintains remote memory[END_REF].

In rodent, neonatal maternal separation (NMS) attenuates neuron differentiation and can lead to stressinduced behavioral disorders. It results in a decrease in RARα expression concomitant with an increase in the RARα promoter methylation. Dnmt1 was shown to be up-regulated, whereas no change was measured in Dnmt3a and Dnmt3b expression in adult dentate gyrus-derived neural precursor cells (ADP). 5AazadC was capable of increasing the neuronal differentiation and decreasing methylation of RARα [START_REF] Boku | Neonatal Maternal Separation Alters the Capacity of Adult Neural Precursor Cells to Differentiate into Neurons Via Methylation of Retinoic Acid Receptor Gene Promoter[END_REF].

f. Depression

Sadness, low mood, loss of motivation, and low self-esteem characterize pathological depression, which, in the most severe cases, can lead to suicide. For example, 38.000 cases of suicide were reported in 2010 in the USA, which represents more than the number of human beings killed by car accident (34.000) or twice more than victims of homicide (16.000) (Nature Editorials, 2014). About 60% of the subjects who committed suicide had depression or related mood disorders.

Until recently, the only epidrugs used to treat depression were HDACi (Sun, 2013). However, a couple of years ago, DNA methylation was proven to be involved in this disorder, and the environment was shown to play a major role, particularly early-life environmental stress [START_REF] Booij | Looking beyond the DNA sequence: the relevance of DNA methylation processes for the stress-diathesis model of depression[END_REF]. Dnmt3a was identified in a mouse model as regulating emotional behavior (LaPlant, 2010). In patients with severe depression and previous suicidal attempts, BDNF hypermethylation was proposed as a biological marker of suicidal behavior [START_REF] Kang | BDNF promoter methylation and suicidal behavior in depressive patients[END_REF]. Additionally, the gene encoding for P11 protein, a modulator of neuronal function involved in depression, was demonstrated to be hypermethylated in rodent and human depression. Interestingly, after treatment with anti-depressant, such as escitalopram (a serotonin reuptake inhibitor), the methylation of P11 was shown to return to normal [START_REF] Melas | Antidepressant treatment is associated with epigenetic alterations in the promoter of P11 in a genetic model of depression[END_REF].

Hypermethylation of the promoter region of TRKB, encoding for tropomyosin receptor kinase B (TrkB), a BDNF receptor, was also reported in suicide completers accompanied by a decrease of mRNA expression [START_REF] Ernst | Alternative Splicing, Methylation State, and Expression Profile of Tropomyosin-Related Kinase B in the Frontal Cortex of Suicide Completers[END_REF].

Sales et al observed that 5aza, 5azadC and RG108 possessed anti-depressant-like effects ion rats, as increasing the mobility in the tail suspension test and decreasing the immobility time in the forced swimming test [START_REF] Sales | Antidepressant-like effect induced by systemic and intra-hippocampal administration of DNA methylation inhibitors[END_REF]. This was correlated to an increase of BDNF expression level. In a model of chronic ultra-middle stress exposure in mice, representative of a depression-like behavior, zebularine and RG108 reverted the depression-like behavior with an increase in Gdnf expression [START_REF] Uchida | Epigenetic Status of Gdnf in the Ventral Striatum Determines Susceptibility and Adaptation to Daily Stressful Events[END_REF].

g. X-chromosome related diseases and autism disorders

Patients with Rett syndrome, predominantly occurring in women, show a normal development for the first 6-18 months, then progressively loose speech and hand use, associated with seizure and autism.

Rett syndrome is an X-chromosome-related neurological disease caused by a mutation in the MeCP2

(methyl-CpG-binding protein 2) protein [START_REF] Amir | Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2[END_REF][START_REF] Guy | Reversal of Neurological Defects in a Mouse Model of Rett Syndrome[END_REF]. This protein is known to bind methylated DNA, repress transcription, and to be involved in gene regulation via the recruitment of the co-activator CREB1 [START_REF] Lyst | Rett syndrome: a complex disorder with simple roots[END_REF]. Although DNA methylation pattern is described as crucial for MeCP2 recognition, no DNMTi was used to treat Rett syndrome. However, treatment of hippocampal cells of MeCP2 knock-out mice with methyl donor SAM was able to rescue neurotransmission event frequencies and thus to partially compensate MeCP2 loss of function [START_REF] Nelson | Activity-Dependent Suppression of Miniature Neurotransmission through the Regulation of DNA Methylation[END_REF].

Additionally, in X-fragile syndrome, another related autism disorder, an hypermethylation in the promoter region of the FMR1 gene is observed [START_REF] Sutcliffe | DNA methylation represses FMR-1 transcription in fragile X syndrome[END_REF]. The treatment of X-fragile cell lines with 5azadC was reported to slightly re-induce FRM1 expression [START_REF] Tabolacci | Differential epigenetic modifications in the FMR1 gene of the fragile X syndrome after reactivating pharmacological treatments[END_REF].

h. Parkinson's and Alzheimer's diseases

Parkinson's disease (PD) is a CNS disease due to the degeneration of dopamine-producing neurons, and it affects more than 4 million people worldwide. The movement-related symptoms include shaking and dementia and can occur in a later stage of the disease. Hypomethylation is observed in PD patients in the CpG rich island of SNCA, gene coding for α-synuclein [START_REF] Jowaed | Methylation Regulates -Synuclein Expression and Is Decreased in Parkinson's Disease Patients' Brains[END_REF]. α-Synulein is a protein involved in aberrant soluble oligomers that lead to neurons death. The reduced levels of SNCA methylation were shown to result from a "sequestration" of DNMT1 in the cytosol in an α-synucleintransgenic mice and post-mortem PD brains [START_REF] Desplats | α-Synuclein Sequesters Dnmt1 from the Nucleus: A Novel Mechanism for Epigenetic Alterations in Lewy Body Diseases[END_REF]. 5azadC was tested on dopaminergic neurons and resulted in viability decrease and increase of apoptosis associated with an up-regulation of α-synuclein (Wang, 2013b). DNMTi can thus have deleterious effects for PD patients.

Alzheimer's disease (AD) is a neurodegenerative disorder resulting in severe dementia. The methylation profile of AD disease patients is controversial as both hypo-and hypermethylation were reported [START_REF] Coppieters | Epigenetics in Alzheimers Disease: a Focus on DNA Modifications[END_REF]. Nuclear immunostaining of DNMT1 and other component of the methylation complex such as MBD2/3 was significantly diminish in neurons [START_REF] Mastroeni | Epigenetic changes in Alzheimer's disease: Decrements in DNA methylation[END_REF], where a loss of methylation of the amyloïd precursor proteins was observed. A decrease in BACE and PSEN1

(encoding for presenilin 1) methylation was also shown in AD patients and was associated with a potential over-expression of amyloid β-peptides [START_REF] Mastroeni | Epigenetic changes in Alzheimer's disease: Decrements in DNA methylation[END_REF][START_REF] Scarpa | Presenilin 1 gene silencing by S-adenosylmethionine: a treatment for Alzheimer disease?[END_REF]. A cocktail of DNA methylation enhancers, such as folate, is currently in phase III clinical trial (NCT00056225). Moreover, a dramatic global hypermethylation in the gray matter of post-mortem human brain tissues was also observed in AD patients [START_REF] Coppieters | Global changes in DNA methylation and hydroxymethylation in Alzheimer's disease human brain[END_REF] together with the increase of methylation in certain regions such as MCF2L and ANK1 genes [START_REF] Jager | Alzheimer's disease: early alterations in brain DNA methylation at ANK1, BIN1, RHBDF2 and other loci[END_REF]. Additional data are crucial to confirm the hypermethylation profile of AD patient and to potentially envisage the use of DNMTi for AD treatment.

i. Aging-related senescence and amyotrophic lateral sclerosis

In amyotrophic lateral sclerosis (ALS), motor neurons death leads to muscle weakness and evolves to breathing difficulties. This neurodegenerative disease, often associated with aging, has a 3-5-year survival prognostic [START_REF] Cleveland | From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS[END_REF]. In motor cortex tissues and spinal cord motor neurons of ALS patients, levels of DNMT1 and DNMT3A were shown to be increased [START_REF] Chestnut | Epigenetic Regulation of Motor Neuron Cell Death through DNA Methylation[END_REF][START_REF] Cho | Bone Marrow-Derived Stromal Cells from Amyotrophic Lateral Sclerosis Patients Have Diminished Stem Cell Capacity[END_REF].

Oh et al. reported an increase in DNMT1 and DNMT3A expression in ALS mesenchymal stromal cells (ALS-MSCs) compared to normal MSCs. RG108 treatment of ALS-MSCs isolated form ALS patients' bone marrow induced anti-senescence factors (TERT, VEGF, ANG), together with the downregulation of senescence factors (ATM, P21). Additionally, a significant improvement in ALS-MSC migration and their differentiation into neurons was observed (Oh, 2015b). Thus, RG108 treatment is promising for a more efficient ALS treatment by autologous cells therapies.

In addition, Oh et al. evaluated the effect of RG108 on human bone marrow mesenchymal stromal cells (hBM-MSCs), which are used in cell therapies. They confirmed the anti-senescence effect of RG108 in in vitro cultures with an increase in the expression of anti-senescence factors (TERT, VEGF, bFGF and ANG) and a decrease of senescence-related factors (ATM, P21 and P53) (Oh, 2015a). Thus, an optimized dose of RG108 (5 µM), for which cell viability is maintained, could greatly improve hBM-MSC potency, which could constitute a real progress in the improvement of the stem cell therapies.

j. Neuronal stem cell

Neuronal stem cell (NSC) cultures are potential sources of transplantable cells to treat neurodegenerative diseases. DNA methylation was proven to be essential for NSC differentiation and proliferation. Folic acid was shown to stimulate neonatal rat NSC proliferation in vitro (Li, 2013), whereas zebularine treatment resulted in the attenuation of their proliferation (Lin, 2014, Luo, 2013) and a reduction of their migration (Singh, 2009b). Hence DNMTi are deleterious for NSC growth, but the use of DNMT activators can provide an interesting way to optimize NSC cultures.

DNA methylation was shown to be implicated in many neurological diseases and psychiatric disorders.

The consequences of DNA methylation depend on the disease and the use of DNMTi can be, in a few cases, unfavorable, like in Alzheimer's disease, but it can also have a positive outcome in pathologies like schizophrenia, depression and post-traumatic stress disorders. Despite the high interest in controlling DNMT activity, the path to use DNMTi as drugs to treat neurological diseases and psychiatric disorders is still long. First, it is made difficult by the lack of animal models to study these pathologies. Second, active molecules must cross the blood-brain barrier, and, for example, 5aza or zebularine are not able to do so. Additionally, toxicity is not acceptable for psychiatric diseases, which often require chronic and life-long treatment. Finally, the specificity of action is also an issue as DNA methylation is involved in biological processes in all tissues and organs. Despite these limitations, the better comprehension of DNA methylation in neurological diseases will led to new therapeutic strategies and new small molecule DNMTi are urgently needed for neurodegenerative diseases and psychiatric disorders [START_REF] Szyf | Prospects for the development of epigenetic drugs for CNS conditions[END_REF].

DNMTi application in cardiovascular diseases

Cardiovascular diseases (CVDs) are the leading cause of death in most of the developed countries and environmental factors such as diet, smoking habits or impaired metabolism are critical risk factors.

Therefore, it is not surprising that epigenetic modifications, including DNA methylation, are involved in these pathologies.

DNA methylation patterns were reported to be altered in several CVDs. In hypertension, the hypermethylation of the HSD11B2 promoter, a gene affecting blood pressure, was observed [START_REF] Friso | Epigenetic control of 11 beta-hydroxysteroid dehydrogenase 2 gene promoter is related to human hypertension[END_REF]. Hypermethylation was also reported for the ATP-binding cassette transporter A1 gene (ABCA1) in coronary heart disease [START_REF] Guay | Acetylsalicylic acid, aging and coronary artery disease are associated with ABCA1 DNA methylation in men[END_REF] and, in an atherosclerosis Apoe -/-mouse model, the hypermethylation of specific vascular homeostasis genes was described [START_REF] Zaina | DNA Methylation Map of Human Atherosclerosis[END_REF]. A study on about 300 Singapore Chinese subjects also proposed DNA methylation as CVD risk biomarker, specifically the Alu/STAT2 methylation level (Kim, 2010). In parallel, in the early-stage atherosclerosis Apoe -/-mouse model a global hypomethylation was reported in peripheral blood mononuclear cells [START_REF] Lund | DNA Methylation Polymorphisms Precede Any Histological Sign of Atherosclerosis in Mice Lacking Apolipoprotein E[END_REF]. Concerning the use of DNMTi in CVDs, in the context of norepinephrine-induced heart hypertrophy, a 6-days treatment with 5azadC restored a normal protein expression profile of the whole cardiac proteome and rescued the phenotype of norepinephrine-treated rats [START_REF] Xiao | Inhibition of DNA methylation reverses norepinephrine-induced cardiac hypertrophy in rats[END_REF]. 5azadC was also shown to facilitated inorganic phosphorus mineralization of human aortic smooth muscle cells (HASMCs) via upregulation of the alkaline phosphatase (ALP), a risk factor for cardiovascular disease in patients with chronic kidney disease (CKD) [START_REF] Azechi | 5-aza-2'-Deoxycytidine, a DNA methyltransferase inhibitor, facilitates the inorganic phosphorus-induced mineralization of vascular smooth muscle cells[END_REF].

Atherosclerosis is characterized by an inflammation of arterial wall and it is the major cause of stroke and heart attack. Gene expression in endothelial cells changes dramatically when submitted to blood flow variations and disturbed flow is pro-arterogenic. In mice, disturbed blood flow induces Dnmt expression, moreover several down-regulated mechano-sensitive genes were identified and their expression was shown to be under their promoter methylation control and reversed by 5azadC [START_REF] Dunn | Flow-dependent epigenetic DNA methylation regulates endothelial gene expression and atherosclerosis[END_REF]. Besides, estrogen receptors (ERα, ERβ) are arteroprotective and hypermethylation of ESR1

(ERα) and ESR2 (ERβ) promoters, associated with their silencing, were shown in atherosclerostic tissues and in senescing cells. Treatment with 5azadC was carried out on smooth muscle cells (SMCs)

and endothelial cells (ECs), resulting in the decrease of estrogen receptor gene promoter methylation accompanied by an increase of ER levels in both vascular cell lines. Additionally, the combination 5azadC/TSA showed some synergetic effect, while TSA alone had no effect (Kim, 2007).

Ischemia is a decrease in blood supply to tissue, which results in hypoxia and leads to cardiac fibrosis.

In hypoxia-induced pro-fibrotic state of human cardiac fibroblast, an increase in DNMT1 and DNMT3B expression and a global hypermethylation were reported. The expression of pro-fibrotic genes, such as alpha-smooth muscle actin (ASMA) or collagen 1, increased and this was enhanced by treatment with TGFβ, a pro-fibrotic cytokine. Interestingly, treatment with 5azadC significantly reduced TGFβ effects and levels of ASMA and collagen 1, decreasing pro-fibrotic effects and positioning DNMT as potentially valuable therapeutic target in ischemic heart disease [START_REF] Watson | Hypoxia-induced epigenetic modifications are associated with cardiac tissue fibrosis and the development of a myofibroblast-like phenotype[END_REF].

Finally, exploring the influence of DNMTi on mouse embryonic stem cells (mESCs), zebularine was shown to re-express cardiac-specific genes, such as Nkx2.5 and Gata4 [START_REF] Horrillo | Zebularine regulates early stages of mESC differentiation: effect on cardiac commitment[END_REF].

DNA methylation is also implicated in heart failure. Indeed, a higher level of homeobox gene PITX2c

promoter methylation accompanied with increased DNMT1 and decreased PITX2 protein levels was identified in heart disease atrium. In the HL-1 cell line, 5azadC reversed PITX2c promoter methylation and increased the PITX2 and KIR2.1 protein level [START_REF] Kao | Heart failure and angiotensin II modulate atrial Pitx2c promotor methylation[END_REF], which could have a positive outcome in case of heart failure.

Recently, DNA methylation and expression levels of COX2 in human heart mesenchymal stem cells (HMSCs) were shown to be involved in the senescence and ageing of HMSCs. Treatment with 5azadC lowered COX2 methylation level, restored its expression level, promoting HMSCs proliferation and delating ageing (Sun, 2021b). This highlighted the potential role of COX2 as a new therapeutic target against cardiovascular diseases. Despite the evidence of the role of DNA methylation in CVDs [START_REF] Chaturvedi | Epigenetic mechanisms underlying cardiac degeneration and regeneration[END_REF], no epidrug is currently in clinical trial for CVDs.

DNMTi application in other human pathologies a. Obesity

Obesity has a high prevalence in industrialized countries where high caloric diet is common. This major public health problem can lead to diabetes or cardiovascular diseases. Following a 5 days high fat diet, DNA methylation changes of the transcriptional co-activator PGC-1α involved in oxidative energy metabolism were observed [START_REF] Brøns | Deoxyribonucleic Acid Methylation and Gene Expression of PPARGC1A in Human Muscle Is Influenced by High-Fat Overfeeding in a Birth-Weight-Dependent Manner[END_REF]. Variations of DNA methylation were also reported by Wang et al., who analyzed DNA methylation in peripheral blood leukocytes and determined that UBASH3A was hypermethylated, whereas TRIM3 was hypomethylated in obesity cases (Wang, 2010b). Variability of DNA methylation in individual adipose tissues was shown to influence their response to caloric restriction in terms of weight loss [START_REF] Bouchard | Differential epigenomic and transcriptomic responses in subcutaneous adipose tissue between low and high responders to caloric restriction[END_REF]. A comparison of methylation levels in abdominal adipose tissues before and after gastric bypass and weight loss showed a decrease of DNA methylation in both tissues after gastric bypass [START_REF] Benton | An analysis of DNA methylation in human adipose tissue reveals differential modification of obesity genes before and after gastric bypass and weight loss[END_REF]. Expression of adiponectin, a protein regulating glucose and lipid metabolism [START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF], was inversely correlated with insulin resistance, type 2 diabetes and cardiovascular diseases [START_REF] Kadowaki | Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome[END_REF]. Kim et al. established that adiponectin expression was under epigenetic control, by hypermethylation of the R2 promoter region, observed in high-fat diet obese mice compared to normal-diet lean mice. RG108 was tested in an obese mice model (db/db mice) and increased adiponectin expression levels were shown to lead to an improvement of glucose intolerance and insulin resistance (Kim, 2015). These recent results are promising for obesity-related disease therapeutics.

b. Alcohol addiction

Alcohol abuse with its associated diseases and behaviors is responsible for about 6% of the death worldwide (WHO 2014 Global status report on alcohol and health). In the United Kingdom, alcohol was classified as the most harmful drug for oneself and others [START_REF] Nutt | Drug harms in the UK: a multicriteria decision analysis[END_REF]. The influence of alcohol consumption on DNA methylation is controversial and seems to be highly dependent on the studied tissues. For example, DNA in post-mortem human brains was reported to be hypomethylated in alcoholics [START_REF] Ponomarev | Gene Coexpression Networks in Human Brain Identify Epigenetic Modifications in Alcohol Dependence[END_REF]. This hypomethylation was shown to result in the increased expression of long-term repeat-containing human endogenous retrovirus. A decrease in DNMT3A and DNMT3B levels was also shown in post-mortem alcoholic patients brains compared to healthy individuals.

DNMT3B expression was reported to be inversely proportional to blood alcohol concentration, whereas no variation was reported for DNMT1 expression level [START_REF] Bönsch | Lowered DNA methyltransferase (DNMT-3b) mRNA expression is associated with genomic DNA hypermethylation in patients with chronic alcoholism[END_REF]. Genome-wide DNA hypermethylation in lymphoblasts of 165 female subjects showed a correlation of the increase in DNA methylation with the drinking frequency [START_REF] Philibert | The Impact of Recent Alcohol Use on Genome Wide DNA Methylation Signatures[END_REF].

DNMTi were studied for the treatment of alcohol addiction and encouraging outcomes were described.

In an alcohol-exposed rat model submitted to the 2-bottle choice test and treated or not with 5aza, Warnault et al. found that for 5aza-treated animals the rate of alcohol intake was significantly lower compared to non-treated alcohol-dependent rats [START_REF] Warnault | Chromatin remodeling [mdash] a novel strategy to control excessive alcohol drinking[END_REF]. These results also showed that inhibition of DNA methylation enabled to specifically reduce alcohol consumption and preference, but did not influence other rewarding substance intake. DNA methylation was suggested to increase the expression of endogenous factors interfering with alcohol drinking behavior, such as BDNF [START_REF] Logrip | Escalating ethanol intake is associated with altered corticostriatal BDNF expression[END_REF] and GDNF [START_REF] Carnicella | GDNF is a fast-acting potent inhibitor of alcohol consumption and relapse[END_REF] that prevent the escalation from moderate to excessive consumption. Barbier et al. studied long-term behavior in alcohol-conditioned rats after 3 weeks of abstinence and established a causal relationship between DNA methylation, alcohol intake and seeking behavior. They measured an increase in Dnmt1 and no change in Dnmt3A and Dnmt3B levels. Using intracerebroventricular infusion of RG108, they lowered the DNA methylation level in mPFC (medial prefrontal cortex) of alcohol-conditioned rats and, while no change was observed in controls. DNMTi also prevented escalation of alcohol consumption. Seven genes involved in neurotransmission and coding for synaptic proteins were down-regulated in alcohol-dependent rats. Among them, synaptotagmin 2 (Syt2) presented a higher methylation level in alcohol post-dependent rats and a synaptic transmission deregulation was reported in post-dependent mPFC neurons. A Syt2 knockdown resulted in compulsive-like drinking behavior. Interestingly, Syt2 normal methylation level and expression were restored by RG108 treatment [START_REF] Barbier | DNA Methylation in the Medial Prefrontal Cortex Regulates Alcohol-Induced Behavior and Plasticity[END_REF].

Although variations of DNA methylation levels in the brain of alcoholic patients seem to be heterogeneous, the results of DNMTi on preclinical animal models are an interesting starting point for the use of DNMTi to limit alcohol dependence.

c. Inflammation and allergy

Inflammation is a reaction of the immune system to harmful stimuli. In allergy, also known as hypersensitivity (e.g. hay fever, allergic asthma, etc.), inflammation is triggered by an unsuitable immune response to harmless environmental cues. Similarly, in auto-immune diseases, an abnormal immune response is directed towards tissues normally present in the organism. Among the CD4 However, in this study, the authors hypothesized that the increase of INFG promoter methylation might not be the only reason of its lower expression and further investigation are required. In mouse models, Dnmt3a and Dnmt3b were more expressed in Th2 cells compared to Th1 cells, whereas no difference in Dnmt1 expression was observed [START_REF] Yu | DNA methyltransferase 3a limits the expression of interleukin-13 in T helper 2 cells and allergic airway inflammation[END_REF]. Moreover, in the same study, INFG and IL4 expression was lower in mutant mice lacking Dnmt3a or Dnmt3a/3b but not in mutants lacking only Dnmt3b. Altogether these data highlight the importance of DNA methylation in the regulation of cytokine expression in CD4 + T-cells and in inflammation reactions.

In inflammation, the balance of T-cell sub-type is disturbed with a shift from Th1 towards Th2 response. As a result, the ratio INF vs IL4 production is modified, which can be considered as an indicator of inflammation risk [START_REF] Shahid | Increased Interleukin-4 and Decreased Interferon-γ in Exhaled Breath Condensate of Children with Asthma[END_REF]. In patients with bronchial asthma, T-cell differentiation into Th2 sub-type and IL4 associated cytokine are much higher than in controls [START_REF] Kwon | DNA Methylation and the Expression of IL-4 and IFN-γ Promoter Genes in Patients with Bronchial Asthma[END_REF].

Although genetics predispositions are involved in asthma [START_REF] Lloyd | Regulatory T Cells in Asthma[END_REF], environment such as pollution, cigarette smoke, etc. is also well-known to have a crucial influence, which rises the importance of epi-mutations in asthmatic patients. A comparison of 21 monozygotic adult twins discordant for asthma showed a decrease in INFG and FOXP3 expression in the case of asthma with a greater decrease for cases with second-hand smoke (SHS) during childhood, which was shown to significantly affect INFG and FOXP3 methylation levels [START_REF] Kohli | Secondhand smoke in combination with ambient air pollution exposure is associated with increasedx CpG methylation and decreased expression of IFN- in T effector cells and Foxp3 in T regulatory cells in children[END_REF][START_REF] Runyon | Asthma Discordance in Twins Is Linked to Epigenetic Modifications of T Cells[END_REF]. A similar study with monozygotic twins concluded the importance of DNA methylation of genes involved in immune response associated with psoriasis, like TNFSF11, in CD4 + T-cells [START_REF] Gervin | DNA Methylation and Gene Expression Changes in Monozygotic Twins Discordant for Psoriasis: Identification of Epigenetically Dysregulated Genes[END_REF]. It is important to underline the significance and increased interest in epidemiology studies on the epigenome of monozygotic twins, starting from the seminal paper of [START_REF] Fraga | Epigenetic differences arise during the lifetime of monozygotic twins[END_REF][START_REF] Fraga | Epigenetic differences arise during the lifetime of monozygotic twins[END_REF].

Concerning seasonal allergic rhinitis (SAR), peripheral blood mononuclear cells from SAR patients and healthy controls were challenged with allergen. Interestingly, the DNA methylation profile of SAR patients varied during and outside pollen season [START_REF] Nestor | DNA Methylation Changes Separate Allergic Patients from Healthy Controls and May Reflect Altered CD4+ T-Cell Population Structure[END_REF]. DNMTi were tested in vitro and in vivo for their ability to control inflammation. In asthma, upon allergen sensitization/challenge by ovalbumine treatment a comparison of sensitized and non-sensitized mice showed an increase in INFG promoter methylation correlating with a decrease in INF level. This increase was reversed by 5azadC treatment [START_REF] Brand | DNA methylation of TH1/TH2 cytokine genes affects sensitization and progress of experimental asthma[END_REF].

In acute lung injury (ALI), an inflammatory lung disease mainly caused by sepsis, the combination of 5azadC and TSA reduced mortality level in LPS (lipopolysaccharide)-induced ALI mouse mode with a 80% survival rate [START_REF] Thangavel | Combinatorial Therapy with Acetylation and Methylation Modifiers Attenuates Lung Vascular Hyperpermeability in Endotoxemia-Induced Mouse Inflammatory Lung Injury[END_REF]. Primary bone marrow-derived macrophages of LPS-treated mice expressed higher levels of pro-inflammatory chemokines and cytokines, which were decreased upon 5azadC/TSA treatment, resulting in cell survival increase [START_REF] Thangavel | Epigenetic modifiers reduce inflammation and modulate macrophage phenotype during endotoxemia-induced acute lung injury[END_REF].

Rheumatoid arthritis is a chronic inflammatory auto-immune disease involving synovial inflammation and causing joint pains. In fibroblast-like synoviocytes (FLS) of arthritic rats, the methyl-CpG-binding protein MeCP2 was selectively over-expressed, whereas secreted frizzled-related protein (SFRP4) was down-regulated. A knock-down of MeCP2 resulted in an enhancement of the SFRP4 level. SFRP4 is known to activate the Wnt pathway, which is involved in FSL abnormal proliferation. Treatment of FLS with 5azadC resulted in an increased SFRP4 expression and a decreased cell proliferation [START_REF] Miao | MeCP2 modulates the canonical Wnt pathway activation by targeting SFRP4 in rheumatoid arthritis fibroblast-like synoviocytes in rats[END_REF]. Finally, a strong increase in promoter methylation of Th2 genes (IL4 and IL5) with an opposite effect for Th1 genes (INFG and IL10) was observed in PMBC of children allergic to cow's milk compared to healthy ones [START_REF] Canani | Differences in DNA methylation profile of Th1 and Th2 cytokine genes are associated with tolerance acquisition in children with IgE-mediated cow's milk allergy[END_REF].

These recent studies described above suggest the potential of DNMTi in the development of new antiinflammatory treatment in asthma, acute lung injury and rheumatoid arthritis.

d. Infectious diseases

Viral infections

In 2014, 35 million people were affected by HIV (human immunodeficiency virus)-1 worldwide. Most infected people have a normal quality of life when treated with anti-retrovirus therapy, but an interruption in the treatment can reactivate latent viruses [START_REF] Van Lint | HIV-1 transcription and latency: an update[END_REF]. Efforts have been made to improve the anti-HIV therapies. It was shown that methylation of proviral DNA of HIV [START_REF] Kumar | Epigenetic control of HIV-1 post integration latency: implications for therapy[END_REF] or human T-lymphotropic virus (HTLV) [START_REF] Saggioro | Transcriptional block of HTLV-I LTR by sequence-specific methylation[END_REF], is involved in latency establishment and in their escape from the immune system. In HIV-infected cells, an increase of DNMT1 [START_REF] Mikovits | Infection with Human Immunodeficiency Virus Type 1 Upregulates DNA Methyltransferase, Resulting in De Novo Methylation of the Gamma Interferon (IFN-γ) Promoter and Subsequent Downregulation of IFN-γ Production[END_REF] and DNMT3A/3B levels [START_REF] Chandel | Epigenetic Modulation of Human Podocyte Vitamin D Receptor in HIV Milieu[END_REF] has been observed. However, the influence of DNA methylation is quite controversial and a hypermethylation of HIV-1 5' long terminal regions (LTR)

was reported in latent HIV reservoirs of patients who did not present viremia compared to viremic patients [START_REF] Blazkova | CpG Methylation Controls Reactivation of HIV from Latency[END_REF]. A strategic option could be to activate HIV in their reservoir to kill infected cells, while anti-retrovirals are used to block new infections. In this context, Fernandez et al.

demonstrated that, in association with TNFα known to activate HIV replication, the use of 5azadC activated HIV twice more than the single use of TNFα in J-Lat cells. However, this seems to be cellline specific, as in other cell lines, such as J1.1 and U1, 5azadC inhibited HIV activation [START_REF] Fernandez | Cell line-dependent variability in HIV activation employing DNMT inhibitors[END_REF]. This variability highlights the complexity in the dynamic of DNA methylation and the need of novel chemical inhibitors to probe the role of DNA methylation in different biological contexts.

HIV is also known to be associated with certain cancers such as non-Hodgkin's lymphomas (NHL). In HIV-positive NHL patients, a clear up-regulation of DNMTs was observed. In HIV-positive aggressive B-cell lymphomas, the HIV-TAT protein was shown to be secreted by infected cells and to result in cell proliferation. In this context, an up-regulation of DNMTs was observed and 5azadC

treatment was able to re-express P16 [START_REF] Luzzi | HIV-1 Tat induces DNMT over-expression through microRNA dysregulation in HIV-related non Hodgkin lymphomas[END_REF].

Similarly, human papillomavirus (HPV)-positive lung cancers exhibited high levels of DNMT3B (Lin, 2005) and treatment with 5azadC was able to restore normal levels of E-cadherin expression in the presence of HPV (D 'Costa, 2012). These findings could be exploited to improve the treatment of virus-induced cancers like HIV-positive NHL, papilloma virus-positive cancers or Epstein-Barr viruspositive Burkitt's lymphoma [START_REF] Paschos | Epstein-Barr Virus Latency in B Cells Leads to Epigenetic Repression and CpG Methylation of the Tumour Suppressor Gene Bim[END_REF].

Hepatitis C virus (HCV) was also shown to lower E-cadherin levels and induce an hypermethylation of the E-cadherin promoter, which is known to induce morphological changes, alter cell-cell adhesion and induce epithelial-mesenchymal transition, critical in tumorigenesis [START_REF] Park | Hepatitis C virus represses E-cadherin expression via DNA methylation to induce epithelial to mesenchymal transition in human hepatocytes[END_REF]. DNA methylation was reported to be deregulated in HCV. DNMT activity was shown to be essential for HCV cell infection and the use of 5aza or 5azadC induced a significant decrease in HCV infection (Chen, 2013). Therefore, DNMTs can represent potential targets for HCV treatment.

Bacterial infections

Epigenetic changes regulate the bacteria-specific innate immune response, as described for inflammation (cf. paragraph 17.3.4.c). Alterations of DNMT expression levels were measured in mice provided with E. coli or E. coli-LPS contaminated water compared to control mice. This highlighted the role of bacterial LPS in the alteration of the epigenetic response [START_REF] Kovalchuk | The increased expression of proteins involved in proliferation, DNA repair and DNA methylation in spleen of mice exposed to E. coli O157:H7 lipopolysaccharide[END_REF]. In E. coliinfected human uroepithelial cells, DNMT activity was demonstrated to be more than 10-fold enhanced compared to non-infected cells [START_REF] Tolg | Uropathogenic E. coli infection provokes epigenetic downregulation of CDKN2A (p16INK4A) in uroepithelial cells[END_REF]. DNMT1 expression was also altered following

Porphyromonas gingivalis or Fusobacterium nucleatum infection [START_REF] Yin | Epigenetic regulation of human -defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria[END_REF]. In parallel, pretreatment of gingival epithelial cells with 5aza significantly reduced the up-regulation of cytokine genes IL6 and CXCL1, observed upon P. gingivalis or F. nucleatum exposure. However, no reduction in bacterial invasion of gingival epithelial cells was observed [START_REF] Drury | DNA methylation differentially regulates cytokine secretion in gingival epithelia in response to bacterial challenges[END_REF]. Hence, although DNA methylation is involved in bacterial infection, the use of DNMTi is still to be studied.

Parasite infections

DNA methylation was shown to be involved in several parasite infections. Quinazoline-based DNMTi, show oral efficacy in P. berghei infected mice [START_REF] Bouchut | Identification of novel quinazoline derivatives as potent antiplasmodial agents[END_REF] and quinoline-quinazoline DNMTi induced parasite death including artemisinin-resistant pathogens together with a decrease in methylation level [START_REF] Nardella | DNA Methylation Bisubstrate Inhibitors Are Fast-Acting Drugs Active against Artemisinin-Resistant Plasmodium falciparum Parasites[END_REF]. DNA methylation inhibition was also studied in Biomphalaria glabrata, a mollusk, host of the pathogen Schistosoma mansoni. In this invertebrate model, 3-bromo-3-nitroflavanones were shown to reduce DNA methylation in B. glabrata and its offspring together with phenotype modification but with no noticeable toxicity. Interestingly, the identified hypomethylated loci were associated with reduced gene expression [START_REF] Luviano | The methylome of Biomphalaria glabrata and other mollusks: enduring modification of epigenetic landscape and phenotypic traits by a new DNA methylation inhibitor[END_REF].

The importance of the epigenetic modulation of the host in case of infective disease starts to be established and this could open the door to the use of epidrugs as anti-infectious agents. This would certainly be facilitated by the discovery of more potent and selective small molecule DNMTi.

e. Embryo growth

The establishment and maintenance of DNA methylation patterns are crucial in the early development of the human embryo [START_REF] Guo | The DNA methylation landscape of human early embryos[END_REF]. Altered DNMT activity in the embryo can lead to early pregnancy loss [START_REF] Yin | Insufficient maintenance DNA methylation is associated with abnormal embryonic development[END_REF]. Early DNA methylation pattern establishment is also important in the in vitro fertilization (IVF) process and development of pre-implanted embryos was shown to be dependent on correct methylation [START_REF] Dobbs | Dynamics of DNA Methylation during Early Development of the Preimplantation Bovine Embryo[END_REF]. To better understand and improve the success rate in IVF process, studies were carried out on DNA methylation pattern in animal model embryos. S-Adenosyl-L-homocysteine (SAH), a common inhibitor of methyltransferase, was used to study in vitro mouse embryo development. Administered at a period of time preceding de novo methylation, SAH results in a decrease of DNA methylation and improvement of development competency of in vitro cloned embryos compared to non-treated ones [START_REF] Jafari | Improved in vitro development of cloned bovine embryos using S-adenosylhomocysteine, a non-toxic epigenetic modifying reagent[END_REF]. Treatment of buffalo skin fibroblast donor cells with the combination 5azadC/TSA decreased global DNA methylation, apoptosis and improved the development of cloned embryos [START_REF] Saini | Treatment of buffalo (Bubalus bubalis) donor cells with trichostatin A and 5-aza-2'-deoxycytidine alters their growth characteristics, gene expression and epigenetic status and improves the in vitro developmental competence, quality and epigenetic status of cloned embryos[END_REF]. RG108 in combination with an HDAC inhibitor showed synergetic effects on somatic cell nuclear transfer, a technique that is known to have limited efficiency in mammalian cloning and in which the DNA methylation profile was shown to be incompletely reprogrammed [START_REF] Peat | Incomplete methylation reprogramming in SCNT embryos[END_REF]. RG108 at high doses (500 µM) increased embryoblasts compared to non-treated controls, which could be promising for the improvement in cloning efficiency [START_REF] Li | Effect of DNA Methyltransferase Inhibitor, RG108, on In Vitro Development and ntES Establishment Rate in Cloned Mouse Embryos[END_REF][START_REF] Watanabe | Epigenetic regulation in pluripotent stem cells: a key to breaking the epigenetic barrier[END_REF].

DNMTi application in metabolite production

To date, no direct therapeutic studies targeting DNA methylation in fungal infection were carried out.

However, DNA methylation was found to be involved in fungal biosynthetic genes repression. When cultured in the presence of 50 µM of 5aza, atlantic-forest-soil-derived Penicillium citreonigrum produced fungal exudates, formally known as guttates, secondary metabolites strongly enriched in several specific components, as the azaphilone family, and exhibited new secondary metabolites from the atlantinone family (Wang, 2010a). Likewise, supplementation of the Alternaria sp. fungus culture medium by 5aza induced the production of toxic metabolites, such as alternariol, altenusin, alternariol-5-O-methyl ether, 3'-hydroxyalternariol-5-O-methyl ether, which are known as plant-disease inducers and that are dormant in normal culture conditions [START_REF] Sun | Induced production of mycotoxins in an endophytic fungus from the medicinal plant Datura stramonium L[END_REF].

Additionally, DNMTi and HDACi can be added to bacterial cultures to diversify the metabolite production. For example, 5aza was added to marine fungus Leucostoma perisoonii culture medium to optimize the production of cytosporones; two unknown cytosporones were obtained, one of which exhibiting some activity against Plasmodium falciparum [START_REF] Beau | Epigenetic Tailoring for the Production of Anti-Infective Cytosporones from the Marine Fungus Leucostoma persoonii[END_REF]. This modulation of the biosynthesis pathways through DNA methylation tailoring could lead to the production and identification of novel molecules that can be therapeutically active compounds

DNMTi in plants

Variations in plant growth conditions, such as temperature, induce epimutations, which can lead to significant modifications in the phenotype, as for example the variation of petal number of the Rosa hybrid. This modification was related to DNA hypermethylation of the RhAG promoter at low temperature [START_REF] Cortijo | Mapping the Epigenetic Basis of Complex Traits[END_REF][START_REF] Ma | Low temperature-induced DNA hypermethylation attenuates expression of RhAG, an AGAMOUS homolog, and increases petal number in rose (Rosa hybrida)[END_REF]. In contrast to humans, cytosine methylation in plants does not occur only in a CpG context and 30% of the cytosines are methylated compared to 5-8% in humans [START_REF] Finnegan | DNA Methylation in Plants[END_REF]. In Japonica rice (Oryza sativa L.), 5aza or 5azadC (0.3 mM for 16 h to 3 days) were

shown to decrease the DNA methylation level and to result in dwarfism. The effect of 5azadC (70% dwarfism effect) was more pronounced than with 5aza (30% dwarfism effect). Unlike in mammals, the DNA methylation profile is not erased during gametogenesis [START_REF] Kinoshita | One-Way Control of FWA Imprinting in Arabidopsis Endosperm by DNA Methylation[END_REF][START_REF] Kinoshita | Control of FWA gene silencing in Arabidopsis thaliana by SINE-related direct repeats[END_REF], which results in heritable DNA methylation changes in progenies, thus 5aza induced dwarfism in rice was shown to be maintained up to the third generation [START_REF] Sano | A single treatment of rice seedlings with 5azacytidine induces heritable dwarfism and undermethylation of genomic DNA[END_REF]. Upon treatment with 5azadC, a lower methylation at the Xa21G promoter was measured. Xa21G is a resistance gene to the pathogenic bacterium, X. oryzae pv. Oryzae [START_REF] Ronald | The molecular basis of disease resistance in rice[END_REF], and, indeed, the new rice line obtained upon 5aza treatment was proven to possess a resistance trait towards bacterial infection [START_REF] Akimoto | Epigenetic Inheritance in Rice Plants[END_REF].

Thus, DNMTi can program new features in plants and cultures, like the disease-resistance trait described above or flowering [START_REF] Kondo | Induction of flowering by 5-azacytidine in some plant species: relationship between the stability of photoperiodically induced flowering and flower-inducing effect of DNA demethylation[END_REF]. However, as long as no plant specific DNMTi is identified, attention must be taken that these treatments do not to affect the methylation profile of animals and human beings.

Innovative indirect and combined approaches to better target DNA methylation

Since direct targeting of DNMTs is highly challenging other approaches can be envisaged. Rather that targeting DNA methylation writers, DNA methylation readers responsible for sensing the presence of methylated DNA and triggering signaling cascades can also be considered. Besides, DNMTi can also be coupled with other active moiety to lead to bifunctional inhibitors (Figure 17.4).

Methyl-CpG binding proteins: nature and probes

In addition to the writers of DNA methylation, the DNMT enzymes, and the erasers, the TET enzymes, the readers proteins, known as Methyl-CpG Binding Proteins (MBPs), recognize and bind to methylated cytosine and transform this modification into a biological signal [START_REF] Ludwig | Modifiers and Readers of DNA Modifications and Their Impact on Genome Structure, Expression, and Stability in Disease[END_REF]. As DNA methylation is involved in numerous biological processes and in diseases, the MBPs play a global role as well [START_REF] Sidhu | UHRF1: The key regulator of epigenetics and molecular target for cancer therapeutics[END_REF][START_REF] Stirzaker | Methyl-CpG-binding protein MBD2 plays a key role in maintenance and spread of DNA methylation at CpG islands and shores in cancer[END_REF][START_REF] Van Roy | A role for Kaiso-p120ctn complexes in cancer?[END_REF]. Indeed, abnormal MBPs expressions and structures have been reported in psychiatric disorders [START_REF] Gigek | Methyl-CpG-Binding Protein (MBD) Family: Epigenomic Read-Outs Functions and Roles in Tumorigenesis and Psychiatric Diseases[END_REF], in different cancer types [START_REF] Gigek | Methyl-CpG-Binding Protein (MBD) Family: Epigenomic Read-Outs Functions and Roles in Tumorigenesis and Psychiatric Diseases[END_REF], Li, 2020b, Prokhortchouk, 2006) and in inflammatory diseases [START_REF] Robinson | Kaiso-induced intestinal inflammation is preceded by diminished E-cadherin expression and intestinal integrity[END_REF], for example.

Three different subfamilies of MBPs are known at the date: Methyl-CpG Binding Domain Proteins (MBDs), the SET and RING Associated (SRA) proteins and the Kaiso proteins [START_REF] Sidhu | UHRF1: The key regulator of epigenetics and molecular target for cancer therapeutics[END_REF].

a. Methyl-CpG binding domain proteins (MBD)

The Methyl-CpG Binding Domain Proteins (MBD) subfamily is the largest group of MBPs with seven members in total: MeCP2 [START_REF] Lewis | Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA[END_REF], MBD1 [START_REF] Fujita | Methylation-mediated transcriptional silencing in euchromatin by methyl-CpG binding protein MBD1 isoforms[END_REF], MBD2 [START_REF] Hendrich | Identification and characterization of a family of mammalian methyl-CpG binding proteins[END_REF], MBD3 [START_REF] Hendrich | Identification and characterization of a family of mammalian methyl-CpG binding proteins[END_REF], MBD4 [START_REF] Hendrich | Identification and characterization of a family of mammalian methyl-CpG binding proteins[END_REF], MBD5 [START_REF] Baymaz | MBD5 and MBD6 interact with the human PR-DUB complex through their methyl-CpG-binding domain[END_REF], MBD6 [START_REF] Baymaz | MBD5 and MBD6 interact with the human PR-DUB complex through their methyl-CpG-binding domain[END_REF]. All of them are characterized by an MBD domain comprising approximately 80 amino acids, which mediates their binding to methylated DNA (except for MBD3). MBD4 has in addition an enzymatic activity through its glycosylase domain [START_REF] Hendrich | The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites[END_REF]. Furthermore, four other proteins possess an MBD binding domain in their structure [START_REF] Du | Methyl-CpG-binding domain proteins: readers of the epigenome[END_REF]: SETDB1 and SETDB2, which also have a SET domain bearing an histone methyltransferase activity [START_REF] Falandry | CLLD8/KMT1F is a lysine methyltransferase that is important for chromosome segregation[END_REF][START_REF] Schultz | SETDB1: a novel KAP-1associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins[END_REF], BAZ2A (also known as TIPS5) and BAZ2B, which have a bromodomain, explaining their affinity for acetylated histones [START_REF] Jones | A Novel Family of Bromodomain Genes[END_REF].

The MBD proteins are involved in several protein regulatory complexes, such as the NuRD complex, which mainly suppresses genes transcription [START_REF] Xue | NURD, a novel complex with both ATPdependent chromatin-remodeling and histone deacetylase activities[END_REF]. Because of their role in mediating the DNA methylation signal, they constitute an interesting target for chemical modulation, as, for example, the disruption of the binding of the MBD domain to methylated DNA could potentially reactivate silenced genes.

Currently, only three binders of the MBD domain have been identified in the literature, all are targeting MBD2: NF449, aurintricarboxylic acid [START_REF] Wyhs | Time-Resolved Fluorescence Resonance Energy Transfer Assay for Discovery of Small-Molecule Inhibitors of Methyl-CpG Binding Domain Protein 2[END_REF] and KCC-07 (Zhu, 2018) (cf. Figure 17.5). The first two first compounds were identified in an time-resolved (TR)-FRET screening assay for compounds that disrupt the interaction between the MBD domain of hMBD2 and methylated DNA (IC50 of 290 nM for NF449 and 2.7 nM for aurintricarboxilic acid) [START_REF] Tang | A time-resolved fluorescence resonance energy transfer screening assay for discovery of protein-protein interaction modulators[END_REF]. Previously, these compounds were known as a P2X1 receptor antagonist and a well-known inhibitor of multiple DNAprotein interactions, respectively. However, both compounds present features that are not favourable for chemical probes or drugs as they interfere with multiple process, lack complete selectivity and bear reactive moieties [START_REF] El-Ajouz | Molecular basis of selective antagonism of the P2X1 receptor for ATP by NF449 and suramin: contribution of basic amino acids in the cysteine-rich loop[END_REF][START_REF] Roos | Identification of aurintricarboxylic acid as a selective inhibitor of the TWEAK-Fn14 signaling pathway in glioblastoma cells[END_REF][START_REF] Sun | Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase[END_REF]. Compound KCC-07 was previously described to interfere with the binding of MBD2-MBD domain to a methylated 45mer DNA with an IC50 of 1.55 µM (determined by ELISA), while inhibition of MeCP2-DNA binding was undetectable [START_REF] Reichert | Targeting epigenetic repression by interfering with methyl-binding domain protein function[END_REF]. Interestingly, the compound showed in vivo activity extending the lifespan of medulloblastoma xenografted mice by 6 days and restoring Brain-specific angiogenesis inhibitor (BAI1) expression (Zhu, 2018). These results are promising for the use of chemical compounds that target MBDs.

In addition, two compounds have been identified to bind a MBD2 Intrinsic Disordered Domain (IDD) outside the MBD domain: ABA and APC (Kim, 2019) (Figure 17.5). These compounds disrupt the recruitment of P66 by MBD2, which is a key factor of the NuRD complex formation (Gnanapragasam Merlin, 2011) with an IC50 of 1.93 µM for ABA and 1.75 µM for APC (determined by FRET) [START_REF] Schultz | SETDB1: a novel KAP-1associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins[END_REF].

b. SET and RING Associated (SRA) domain Proteins

The SRA proteins are also able to bind methylated and hydroxymethylated DNA, in particular hemimethylated DNA, through the SRA domain. UHRF1 [START_REF] Fujimori | Cloning and mapping of Np95 gene which encodes a novel nuclear protein associated with cell proliferation[END_REF] and UHRF2 [START_REF] Fujimori | Cloning and mapping of Np95 gene which encodes a novel nuclear protein associated with cell proliferation[END_REF] are the two human members of this family. These proteins can recruit epigenetic silencing factors such as DNMT1 and HDAC1 and promote gene silencing [START_REF] Niinuma | UHRF1 depletion and HDAC inhibition reactivate epigenetically silenced genes in colorectal cancer cells[END_REF]. In particular, it has been shown that they play a key role in the recruitment of DNMT1 to hemimethylated DNA [START_REF] Felle | The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1[END_REF]. These proteins act as "pivots" in the gene silencing machinery since they are able to recruit different types of proteins and enzymes to ensure epigenetic DNA methylation, histone methylation and acetylation and even ubiquitin-related actors [START_REF] Choudhry | Targeting microRNA/UHRF1 pathways as a novel strategy for cancer therapy[END_REF].

So far, no binder of UHRF1 and UHRF2 has been identified that disrupt their interaction with methylated DNA. The described inhibitors for UHRF1 target mainly its Tandem Tudor Domain and Plant Homeodomain (TTD and PHD, respectively), which mediate the binding to silencing-related proteins such as DNMT1 (Achour, 2008) and HDAC1 [START_REF] Unoki | ICBP90, an E2F-1 target, recruits HDAC1 and binds to methyl-CpG through its SRA domain[END_REF]. The most relevant molecules are NV03 [START_REF] Senisterra | Discovery of Small-Molecule Antagonists of the H3K9me3 Binding to UHRF1 Tandem Tudor Domain[END_REF], NSC232003 [START_REF] Myrianthopoulos | Tandem virtual screening targeting the SRA domain of UHRF1 identifies a novel chemical tool modulating DNA methylation[END_REF], BPC [START_REF] Houliston | Conformational dynamics of the TTD-PHD histone reader module of the UHRF1 epigenetic regulator reveals multiple histonebinding states, allosteric regulation, and druggability[END_REF] NSC232003, that interacts with UHRF1, decreases the global number of methylated CpGs of a population of U251 glioblastoma cells by 50% [START_REF] Myrianthopoulos | Tandem virtual screening targeting the SRA domain of UHRF1 identifies a novel chemical tool modulating DNA methylation[END_REF]. However, the specificity of these molecules towards UHRF1 over UHRF2 has not been established.

c. Kaiso Proteins

The Kaiso protein subfamily is composed of three different members: Kaiso (Daniel Juliet, 1999), ZBTB4 [START_REF] Filion | A family of human zinc finger proteins that bind methylated DNA and repress transcription[END_REF] and ZTB38 [START_REF] Sasai | Identification of a novel BTB-zinc finger transcriptional repressor, CIBZ, that interacts with CtBP corepressor[END_REF]. These are Zinc Finger Domains (ZF) proteins that bind methylated and unmethylated DNA in a sequence-specific manner [START_REF] Donaldson | Kaiso Represses the Cell Cycle Gene cyclin D1 via Sequence-Specific and Methyl-CpG-Dependent Mechanisms[END_REF]. They are known to recruit HDAC-dependent transcriptional repressors (for ZBTB4 and ZBTB38) [START_REF] Sasai | Identification of a novel BTB-zinc finger transcriptional repressor, CIBZ, that interacts with CtBP corepressor[END_REF], or the N-CoR repressive complex (for Kaiso) [START_REF] Yoon | N-CoR mediates DNA methylation-dependent repression through a methyl CpG binding protein Kaiso[END_REF]. The implication of these proteins in diseases remarkably differs from one another, going from oncogenesis (Buck-Koehntop [START_REF] Bethany | Molecular basis for recognition of methylated and specific DNA sequences by the zinc finger protein Kaiso[END_REF], to abnormal inflammatory mechanisms [START_REF] Ocskó | Transcription factor Zbtb38 downregulates the expression of anti-inflammatory IL1r2 in mouse model of rheumatoid arthritis[END_REF] and even cell cycle dysregulations [START_REF] Blue | Variants regulating ZBTB4 are associated with age-at-onset of Alzheimer's disease[END_REF]. To this day, no inhibitor or probe has been identified, even if in silico screening assays and computational docking simulations have been realized [START_REF] Chikan | KAISO inhibition: an atomic insight[END_REF]. This subfamily is interesting since its members do not share similar biological outcomes [START_REF] Filion | A family of human zinc finger proteins that bind methylated DNA and repress transcription[END_REF] and their depletion in mice has dramatically opposite consequences between Kaiso (viable and fertile) (Prokhortchouk, 2006) and the two other members (cell damage and genomic instability) [START_REF] Marchal | Depletion of ZBTB38 potentiates the effects of DNA demethylating agents in cancer cells via CDKN1C mRNA upregulation[END_REF][START_REF] Roussel-Gervais | Loss of the Methyl-CpG-Binding Protein ZBTB4 Alters Mitotic Checkpoint, Increases Aneuploidy, and Promotes Tumorigenesis[END_REF].

As illustrated, chemical probes for MBPs are still at an early stage with few active molecules identified to date. Mostly they lack specificity and are not potent enough for cellular or in vivo assessments. Nevertheless, these proteins remain an interesting chemical target since they could allow studying complex epigenetic mechanisms and widen the spectra of therapeutic approaches for human pathologies.

Bifunctional inhibitors involving DNMTi

Another strategy to address the lack of efficacy and selectivity of DNMTi is the use of bifunctional compounds. This promising strategy consists in designing bifunctional inhibitors formed by the connection of at least two different pharmacophores in a single drug [START_REF] Tomaselli | Epigenetic polypharmacology: A new frontier for epi-drug discovery[END_REF]. Such compounds have many advantages compared to the use of drug combination. First, selectivity might be improved, pharmacological properties can be modulated and toxicity can be lowered. Besides, both inhibitors are simultaneously present in the same tissue that can help to reach greater efficacy against advanced-stage disease. Bifunctional compounds also limit resistance mechanisms (de Lera, 2016).

Due to the lack of reported potent DNMTi, it is only recently that a few bifunctional inhibitors including a DNMTi moiety have been reported. Synthesis and biological evaluation of four different DNMT/HDAC and DNMT/G9a bifunctional inhibitors have recently been described (Figure 17.7).

Because of the co-location of these two targets in the same protein complex [START_REF] Hervouet | Specific or not specific recruitment of DNMTs for DNA methylation, an epigenetic dilemma[END_REF], their involvement in the same diseases [START_REF] Esteller | Epigenetics in Cancer[END_REF], and the synergic effect of drug combinations, targeting these two enzymes, exhibit good potency. The biggest challenge in designing hybrid inhibitors is to maintain or improve activity against both enzymes.

Histone deacetylases (HDAC) are a family of 18 enzymes, divided in 4 classes, which catalyze deacetylation of histone-tail N-terminal lysines, leading to chromatin compaction [START_REF] Ajmd | Histone deacetylases (HDACs): characterization of the classical HDAC family[END_REF]. In some diseases, particularly in cancer, histone hypoacetylation combined with DNA hypermethylation, cause transcriptional repression [START_REF] Kondo | Epigenetic Cross-Talk between DNA Methylation and Histone Modifications in Human Cancers[END_REF]. HDAC inhibitors have all the same general structure characterized by a Zinc Binding Group (ZBG) that chelate the zincite ion in the catalytic pocket of the enzyme, a hydrophobic linker and a cap group, which interacts at the entrance of the catalytic pocket.

To date, four HDACi were approved: Vorinostat, Belinostat, Panobinostat, and Chidamide, where ZBG is an hydroxamic acid moiety [START_REF] Ho | Thirty Years of HDAC Inhibitors: 2020 Insight and Hindsight[END_REF].

In 2017, the first-in-class bifunctional DNMT/HDAC inhibitor was described [START_REF] Yuan | Design, synthesis and anticancer potential of NSC-319745 hydroxamic acid derivatives as DNMT and HDAC inhibitors[END_REF]. Its design consists in was based on NSC-319745 DNMTi pharmacophore functionalized with an hydroxamic acid moiety. NSC-319745, had been previously identified by virtual screening as a selective DNMT1

inhibitor with low activity (34% inhibition at 100 μM on enzymatic assay) (Kuck, 2010b).

Interestingly, despite its low efficacy, this DNMTi shares similarities with hydroxamate HDAC inhibitors. Changing carboxylic acid in hydroxamic acid, more precisely with a N-hydroxycinnamide moiety, and optimizing its structure allowed to improve the activity against target enzymes. The lead compound finally showed 70%, 23% and 49% inhibition at 100 µM against DNMT1, 3A et 3B, respectively and IC50 at nanomolar range against two classes of HDAC ion enzyme assays. Potent antiproliferative activity was measured in K562 and U937 human cancer cell lines (IC50= 2.9 μM and 1.1 μM, respectively) as well as an increased H3K9 and H4K8 acetylation level. This bifunctional inhibitor was shown to initiate P16 methylation inhibition and its re-expression and to induce apoptosis in leukemia model cancer cells U937.

Another HDAC/DNMT bifunctional inhibitor was described by Rabal's team [START_REF] Rabal | Design and Synthesis of Novel Epigenetic Inhibitors Targeting Histone Deacetylases, DNA Methyltransferase 1, and Lysine Methyltransferase G9a with In Vivo Efficacy in Multiple Myeloma[END_REF]. They first worked on a series of quinoline-based analogue as bifunctional DNMT/G9a inhibitor, where G9a is a lysine methyltransferase. Their hit compound exhibited IC50 of 8 nM against G9a and 382 nM against DNMT1 on purified enzymes (San José-Enériz, 2017) and it was shown to act as a substrate competitive inhibitor of G9a and DNMTs [START_REF] Rabal | Discovery of Reversible DNA Methyltransferase and Lysine Methyltransferase G9a Inhibitors with Antitumoral in Vivo Efficacy[END_REF]. They then designed a multitarget epigenetic inhibitor targeting HDAC, DNMT1 and optionally G9a. Based on structure design, docking and structure activity relationship SAR) analysis, they inserted a ZBG at the solvent exposed area of their inhibitor. They selected the pyrimidylhydroxamic acid moiety of quisininostat since its cap group mimics a quinoline base. Substitution of the quinoline moiety increased activity against DNMT and G9a and finally led to two potent compounds, one acting as a trifunctional G9a/HDAC/DNMT inhibitor and the second one as a bifunctional DNMT/HDAC inhibitor obtained by removing the lysine mimic moiety. Although they lost efficacy against DNMT1, compared with their previous hit compounds, these multifunctional compounds exhibited IC50 in nanomolar range against HDAC1, 2 and 3 and a potent antiproliferative activity on MM.1S multiple myeloma cell line. Due to their poor pharmacological property profiles these compounds should be optimized to enable in vivo experiments.

By fragment-based rational drug design, Jiang's group worked on a series of hydroxamic acid derivatives of nucleoside bases. This work was inspired by cytosine and hydroxamic acid derivatives, FDA-approved DNMTi and HDACi, respectively. In both studies, both pharmacophores were linked through a triazole with different linker lengths. The first bifunctional inhibitor reported exhibiteds adenine as DNMT pharmacophore [START_REF] Ren | Combined inhibition of HDAC and DNMT1 induces p85α/MEKmediated cell cycle arrest by dual target inhibitor 208 in U937 cells[END_REF], then they completed the series varying the nucleobase (Sun, 2021a). The most potent compound, carrying a cytosine linked to the hydroxamic acid moiety with a linear six-carbon alkyl chain showed potent inhibitory activity against DNMT1 with IC50= 6.39 μM and nanomolar activities against HDAC1 and HDAC6, comparable to Vorinostat. Western Blot in U937 lymphoma cell lines treated with this DNMT-HDAC inhibitor, showed an increase of H3K9 and H4K8 acetylation levels and a weak decrease of methylation levels in P16 promoter region that lead to an increase of P16 expression and anti-proliferative activity in a dose-dependent manner. Even though these bifunctional inhibitors displayed a good potency, it they need further investigations for cancer therapy, especially regarding quantification of DNA methylation inhibition in cells, pharmacological properties as well as comparison with DNMTi and HDACi combinations.

The most potent bifunctional inhibitor known to date was described by [START_REF] Yuan | Development of a versatile DNMT and HDAC inhibitor C02S modulating multiple cancer hallmarks for breast cancer therapy[END_REF]. In this study, DC-517, a selective DNMT1 inhibitor described by Luo's group composed of two carbazole groups and a side chain (Chen, 2014) In red the DNMT pharmacophore, in blue the HDAC pharmacophore, in green the G9a pharmacophore, in purple, merged part of pharmacophores

Limits and hopes of DNMTi applications and new perspectives

The above examples illustrate the major role of DNMTs in the normal and abnormal functioning of cells and how their inhibition by DNMTi can change phenotypes, revealing the high potential of DNMTi, both for therapeutic strategies and for the bioengineering of organisms. It also highlights how, depending on the context, the effects can be inversed. This is related to the dynamics of DNA methylation and its role in controlling gene expression. As illustrated by the above examples, DNMTi are just starting to be applied to diseases other than cancer and these strategies seem very promising.

An increasing number of clinical trials are starting to explore epigenetic reprogramming by DNMTi in combination with other treatments, in particular, in solid tumors. Time will show if these studies can validate the interest of epidrugs beyond hematological cancers, the only diseases they are approved for.

Noteworthy, the only FDA-approved DNMTi are 5aza and 5azadC, suicide substrates that are incorporated into DNA and form an irreversible covalent complex with the enzyme. This triggers a subsequent DNMT degradation by the proteasome, inducing a potent inhibition of DNA methylation.

It is not clear whether a non-covalent inhibitor can induce this level of demethylation [START_REF] Erdmann | Targeting DNA methylation with small molecules: what's next?[END_REF].

To obtain the same potency as covalent nucleoside inhibitors, non-nucleoside inhibitors could be an alternative, but they would need to be very specific for the DNMTs and today this specificity is lacking. Among non-nucleoside DNMTi, GSK3484862 showed the most promising results with high in vitro and in cell potency. Nevertheless, it is urgent to find novel compounds that are very potent inhibitors of DNMTs and bind strongly to the enzyme, in order to induce a strong inhibition in cells and eventually a down-regulation of the DNMTs.

Another feature of 5aza, 5azadC and their analogues is the fact that they are globally incorporated into the DNA instead of deoxycytosines and thus they are not specific for CpGs or certain genomic regions.

This can hinder their use by inducing non-desired secondary effects. The use of low doses of the drugs + + has diminished these effects, however repeated cycles are necessary for the epigenetic reprogramming, increasing the probability of their appearance. Non-nucleoside inhibitors have the advantage of not needing incorporation into DNA and thus potentially diminishing the side-effects. In conclusions, it is important to pursue the search for new non-nucleoside inhibitors of DNA methylation that are potent, specific for the CpGs and of promoters silencing key genes in pathologies. Several strategies are being explored for the next generation of inhibitors, as allosteric inhibitors, protein-protein ligands and dual inhibitors [START_REF] Erdmann | Structure-Guided Optimization of DNA Methyltransferase Inhibitors[END_REF].

An interesting alternative is to target the proteins that recognize methylated DNA and trigger the signaling pathways that silence DNA (DNA methylation reader proteins). While, histone reader proteins are a well explored target, the chemical targeting of the MBP is still in its infancy.

Finally, hybrid compounds bearing both a DNMTi moiety and another inhibitor moiety is also promising. Interestingly, targeting not a single enzyme but cancer specific protein complexes can enable cancer cell selectivity. In addition, the merging of two drugs in the same molecules can facilitate the pharmacology compared to drug combinations. Challenges remain in the design of such compounds to maintain both scaffold activity and efficacy.

LEGEND of FIGURES and TABLES

(

  Figure 17.1 top), then transfer of the methyl group from the SAM occurs on the C5 position of the cytosine (Figure 17.1, grey arrow), the cysteine is released by -elimination and the enzyme is ready to start a new catalytic cycle (Figure 17.1 top).

Figure 17 . 1 .

 171 Figure 17.1. Schematic representation of DNMT catalytic cycle mechanism of inhibition by 5azadC.

  with both nucleoside DNMTi (5aza and 5azadC), are currently ongoing, mainly in hematologic cancers with 39 phase III/IV active clinical trials. Many phase III/IV trials have been undertaken in the last few years. 5aza and 5azadC are in phase III in combination with "classical" chemotherapies such as doxorubicin, all-trans retinoic acid (ATRA) or cytarabine. Both nucleosides are also in phase III and IV in combination with Venetoclax, a Bcl2 inhibitor, Glasdegib, a Hedgehog pathway inhibitor, Ivosidenib, an IDH1 inhibitor, Eprenetapopt, a P53 reactivator, or the kinase inhibitors Midostaurin, Gilteritinib, Clofarabine, exclusively against hematological cancers, i.e. AML, MDS or CMML.Besides this, several combinations of nucleoside DNMTi with monoclonal antibodies, such as anti-CD47 (Magrolimab), anti-CD3 (Visilizumab), anti-CD123 (Talacotuzumab), anti-TIM-3 (Sabatolimab) or anti-PD-1 (Camrelizumab), have reached phase III and IV. Finally, a combination of 5azadC and Cedazuridine, a cytidine deaminase inhibitor, was approved in 2020 for the treatment of MDS (Inqovi®).

  + Tcells (T-helper (Th) cells), T-helper 1 (Th1) express specifically interferon- (INF) (Brand, 2012), whereas T-helper 2 (Th2) express interleukin 4 (IL4), IL5 and IL13. The INF (Winders, 2004) and IL4 (Kwon, 2008) cytokine expression is under DNA methylation control and differentiation of naïve T-cell into Th1 or Th2 cells results in a modification of the methylation levels of CpG sites in INFG coding for INF[START_REF] White | CpG methylation patterns in the IFNγ promoter in naive T cells: Variations during Th1 and Th2 differentiation and between atopics and non-atopics[END_REF]. The level of INFG promoter methylation in CD4+ lymphocytes of adults and children with asthma was shown to depend on age, sex and tissues (Lovinsky-Desir, 2014).

Figure 17 . 4 .

 174 Figure 17.4. Alternative strategies to target DNA methylation by either 1) designing bifunctional inhibitor including a DNMTi and another active moiety and 2) targeting DNA methylation reader proteins, i.e. MBPs.

Figure 17 . 5 .

 175 Figure 17.5. MBD2 targeting compounds. KCC-07 interfers with the binding of MBD2-MBD domain to DNA with an IC50 = 1.55 µM (measured by ELISA), NF449 and aurintricarboxylic acid inhibit the interaction of MBD2 with DNA with an IC50 = 290 nM and 2.7 nM , respectively (measured by TR-FRET). ABA and APC bind to IDRs of MBD2 with IC50 = 1.93 and 1.75 µM, respectively (measured by FRET).

  and 2,4lutidine[START_REF] Chang | Discovery of small molecules targeting the tandem tudor domain of the epigenetic factor UHRF1 using fragment-based ligand discovery[END_REF] (Figure 17.6) with an affinity to the TTD domain of Kd = 2 µM for NV03 (determined by ITC and Fluorescence polarisation)[START_REF] Senisterra | Discovery of Small-Molecule Antagonists of the H3K9me3 Binding to UHRF1 Tandem Tudor Domain[END_REF]; 50 µM for BPC (determined by ITC and DSF)[START_REF] Houliston | Conformational dynamics of the TTD-PHD histone reader module of the UHRF1 epigenetic regulator reveals multiple histonebinding states, allosteric regulation, and druggability[END_REF] and 29 µM for 1,4-lutidine (measured by TR-FRET)[START_REF] Chang | Discovery of small molecules targeting the tandem tudor domain of the epigenetic factor UHRF1 using fragment-based ligand discovery[END_REF].

Figure 17 . 6 .

 176 Figure 17.6. UHRF1 binders. BPC, NV03 and 1,4-lutidine bind the UHRF1 Tandem Tudor Domain with Kd= 2 µM, 50 µM and 29 µM, respectively. NSC232003 reduced by 50% global numbers of methylated CpGs in U251 glioblastoma cells.

  Figure 17.7. Bifunctional epigenetic inhibitors containing DNMTi moities.

Figure 17 . 1 .

 171 Figure 17.1. Schematic representation of DNMT catalytic cycle mechanism and its inhibition by 5azadC.

Figure 17 . 2 .

 172 Figure 17.2. Selection of compounds described as DNMT inhibitors.

Figure 17 . 3 .

 173 Figure 17.3. Schematic representation of the mode of action of DNMTi as gene re-expressing agents and examples of DNA methylation-induced gene silencing in diverse pathologies.

Figure 17 . 4 .

 174 Figure 17.4. Alternative strategies to target DNA methylation.

Figure 17 . 5 .

 175 Figure 17.5. MBD2 targeting compounds.

Figure 17 . 6 .

 176 Figure 17.6. UHRF1 binders.

Figure 17

 17 Figure 17.7. Bifunctional epigenetic inhibitors containing DNMTi moities.

5-aza-2'-deoxycytidine 2'-deoxycytidine PHOSPHORYLATION DNA INCORPORATION DNA METHYLATION

	Covalent binding	Methyl group transfer	β-elimination	Enzyme release
			DNMT Inhibition

Table 1 . Examples of DNMTi FDA-approved and in phase II and III clinical trials as a single drug or in combination Phase of clinical trials are mentioned in brackets. Status Drugs Type of cancer targeted (phase of study) as a single agent FDA-approved 5aza (Vidaza) AML, MDS 5azadC (Dacogen) AML, MDS, CMML

 1

	. As

Table 17

 17 .1. Examples of DNMTi FDA-approved and in phase II and III clinical trials as a single drug or in combination

ABBREVIATIONS