WO2019197024A1

WO2019197024A1 - A method of modulating the rna methylation

Info

Publication number: WO2019197024A1
Application number: PCT/EP2018/059284
Authority: WO
Inventors: Simona SELBERG; Daria BLOKHINA; Esko Kankuri; Mati Karelson
Original assignee: Chemestmed Ltd.
Priority date: 2018-04-11
Filing date: 2018-04-11
Publication date: 2019-10-17
Also published as: EP3589745A1

Abstract

Methods and compounds are reported that specifically modulate RNA methylation by activation of the RNA methyltransferase complex METTL3/METTL14. In some embodiments, the compound has binding and/or activation for a METTL3/METTL14/WTAP complex.

Description

A METHOD OF MODULATING THE RNA M ETHYLATION

Technical Field

The presently disclosed subject matter generally relates to the epitrancriptomic regulation of ribonucleic acid (RNA) methylation through small-molecule activators.

Background Art

Chemical modifications of RNA have recently been identified to have an impact on several critical cellular functions, such as proliferation, survival and differentiation, mostly through regulation of RNA stability (Motorin et al., 2017)[9] . The most abundant modification in eukaryotic messenger RNA is N6-methyladenosine (m6A) (Roundtree et al., 2017)[21 ] . It has been shown that m6A modifications of RNA affect its splicing, intracellular distribution, translation, and cytoplasmic degradation, playing thus a crucial role in regulating cell differentiation, neuronal signaling, carcinogenesis and immune tolerance (Maity et al., 2016)[17] . The m6A presence in RNA is regulated by specific enzymes, i.e. the RNA methyltransferases, RNA methylases and RNA reader proteins.

The N-methylation of the adenosine is a reversible process, catalysed by specific enzymes (Figure 1 ). Those include the RNA methyltransferase enzyme complex METTL3/METTL14/WTAP consisting of three components: METTL3

(methyltransferase-like 3), METTL14 (methyltransferase-like 14), and WTAP (Wilm's tumour-1 -associated protein), called also the“writer” enzyme; the RNA demethylases FTO (fat mass and obesity-associated protein) and AlkBH5 (AlkB family member 5), called“erasers”. The fate of the RNA in post-transcriptomic processes is also directed by the“reader” enzymes that recognize specific m6A methylation in RNA. Several RNA reader enzymes have been identified, including YTHDF1 (YTH N6-Methyladenosine RNA Binding Protein 1 ), YTHDF2 (YTH N6-Methyladenosine RNA Binding Protein 2) YTHDF3 (YTH N6-Methyladenosine RNA Binding Protein 3), YTH DC 1 (YTH domain- containing protein 1 ) and YTHDC2 (YTH domain-containing protein 2) (Park et al., 2017)[20]. These three types of enzymes collectively coordinate the m6A RNA methylome in the eukaryotic cell.

The proteins METTL3 and METTL14 were demonstrated to be the catalytic subunits, whereas WTAP controls the process (Sledz et al., 2016)[24]. The hypothetic chemical mechanism on the nucleic acid m6A methylation has been suggested in the case of DNA methyltransferase DNMT1 (Figure 2).

It has been shown that activity-modifying ligands of METTL3/METTL14/WTAP can act as potential anticancer agents (Deng et al., 2018)[5]. Both overexpression of METTL3 and inhibition of the RNA demethylases FTO and AlkBFI5 suppress the growth and self-renewal of glioblastoma stem cell (GSC) and tumorigenesis (Cui et al., 2017, Zhang et al., 2017)[4] The activator small-molecule compounds of METTL3/METTL14 can therefore be potential anticancer agents against tumors such as glioblastoma. Dysregulation of METTL3 expression has also been implicated in growth control of human lung cancer cells (Lin et al., 2016; Du et al., 2017)[15][6]. Consequently, specific METTL3/METTL14 RNA methyltransferase inhibitors could reduce the proliferation of cancer cells. The role of the m6A methylation in the development of myeloid leukaemia is less understood. It has been shown that FTO, as an m6A demethylase, plays a critical oncogenic role in acute myeloid leukaemia (Li Z et al., 2017)[13]. On the other hand, downregulation of METTL3 results in cell cycle arrest, differentiation of leukemic cells and failure to establish leukaemia in immunodeficient mice (Barbieri et al., 2017)[34] In the case of renal cell carcinoma (RCC), up-regulation of METTL3 significantly suppresses tumor growth in vivo (Li X et al., 2017)[12] Similarly, increasing m6A level considerably suppressed tumor development in the case of cervical cancer both in vitro and in vivo (Wang X et al., 2017)[16]. Thus the reduced m6A level is tightly associated with cancer development and activation of the m6A mRNA methylation is a valid therapeutic target.

The reduction of the RNA methyltransferase activity has been shown to inhibit the expression of some viral genomes (Gokhale et al., 2017)[8]. Recent data generated using HIV-1 as a model system strongly suggest that sites of m6A additions enhance virus replication (Kennedy et al., 2017)[1 1 ]. Likewise, m6A residues in Influenza A virus (IAV) transcripts enhance viral gene expression (Courtney et al., 2017)[3]. Therefore, the inhibition of the METTL3/METTL14 may stop the HIV-1 or IAV virus replication. In contrast, depletion of m6A methyltransferases increases the infectious Hepatitis C virus (HCV) particle production (Gokhale et al., 2016)[7] It has been also shown that knockdown of RNA methyltransferases increases, while silencing demethylases decreases, Zika virus (ZIKV) production (Lichinchi et al., 2016)[14] Thus the activation of METTL3/METTL14 would have the antiviral effect against HCV Zika virus and the respective small-molecule activators are valid antiviral therapeutic agents. Summary of invention

The present invention is related to a method of modulating the RNA methylation at 6- position of adenine (m6A) by effective amount of a compound having binding and/or activation for a METTL3/METTL14/WTAP complex.

Also disclosed are the compounds, or salts or esters thereof, which can activate the M ETTL3/M ETT114/WTAP complex.

The "summary of invention" heading is not intended to be restrictive or limiting. The invention also includes all aspects described in the detailed description or figures as originally filed. The original claims appended hereto also define aspects that are contemplated as the invention and are incorporated into this summary by reference.

In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. For example, although aspects of the invention may have been described by reference to a genus or a range of values for brevity, it should be understood that each member of the genus and each value or sub-range within the range is intended as an aspect of the invention. Likewise, various aspects and features of the invention can be combined, creating additional aspects which are intended to be within the scope of the invention. Although the applicant(s) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention.

Brief description of Drawings

The present invention is disclosed further with references to accompanying drawings:

FIG. 1 . Dynamic and reversible m6A methylation in RNA (SAM - S-adenosyl-L- methionine; SAH - S-adenosyl-L-homocystein) (Niu, et al., 2013)[19];

FIG. 2. Hypothetic mechanism of the m6A methylation of DNA by DNA

methyltransferase DNMT1 (Scavetta et al., 2000)[23]; FIG. 3. The binding site of SAH;

FIG. 4. The binding site of the compound (III);

FIG. 5. The binding of SAM and compound (VI) during their simultaneous docking to METTL3. There is a close interaction between the sulfur atom of SAM (A) and the carbonyl group of compound (VI) (B);

FIG. 6. Western blot analysis of the FLAG-tagged protein purified from FIEK293 total lysate. Flag-tag purified proteins were probed with anti-Mettl3, anti-MettH 4 and anti- WTAP antibodies. The sizes of the bands correspond to protein sizes: Mettl3-FLAG 65kDa, MettM 4-FLAG 53kDa, WTAP 44kDa.

FIG. 7. The influence of the small-molecule ligands of the METTL3/METTL14/WTAP complex on the substrate RNA methylation. The graphs represent the percentage of the methylation as compared to the reference reaction (no small-molecule ligand added); (a) compound (III); (b) compound (IV); (c) compound (V); (d) compound (VI).

Detailed Description of invention

Disclosed herein are compounds and methods of modulating the RNA methylation through activation of METTL3/METTL14/WTAP complex. In some variations of the invention, the compound is administered in a composition that also includes one or more pharmaceutically acceptable diluents, adjuvants, or carriers.

The compound can be a small molecule. In some embodiments, METTL3/METTL14/WTAP complex activator has a structure of Formula (I),

wherein: R1 and R2 are independently selected from the group consisting of H, alkyl, aryl, aralkyl, acyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, alkylcarbamoyl, and dialkylcarbamoyl, aminoalkyl, aminoalaryl; or a pharmaceutically acceptable salt thereof. In some embodiments, the METTL3/METT14/WTAP complex activator compound has a structure of Formula (II)

wherein R1 and R2 are independently selected from the group consisting of H, alkyl, aryl, alkylenearyl, acyl, alkoxycarbonyl, aryloxycarbonyl, alkylenearyloxycarbonyl, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, and alkyleneamino; In some embodiments, R1 and R2 are independently selected from the group consisting of alkyleneamino and hydrogen, where the amino group of the alkyleneamino moiety can be further substituted with one or two alkyl or alkylenearyl (e.g., a benzyl) groups. In a specific embodiment, R1 is methyl and R2 is hydrogen.

In some embodiments, the METTL3/METTL14/WTAP complex activator compound has a structure of Formula (III)

or a pharmaceutically acceptable salt thereof. In some embodiments, the METTL3/METTL14/WTAP complex activator compound has a structure of Formula (IV)

or a pharmaceutically acceptable salt thereof.

In some embodiments, the METTL3/METTL14/WTAP complex activator compound has a structure of Formula (V)

or a pharmaceutically acceptable salt thereof.

In some embodiments, the METTL3/METTL14/WTAP complex activator compound has a structure of Formula (VI)

or a pharmaceutically acceptable salt thereof.

As used herein, the term "alkyl" refers to straight chained and branched hydrocarbon groups containing carbon atoms, typically methyl, ethyl, and straight chain and branched propyl and butyl groups. Unless otherwise indicated, the hydrocarbon group can contain up to 20 carbon atoms. The term "alkyl" includes "bridged alkyl," i.e., a C.sub.6-C.sub.16 bicyclic or polycyclic hydrocarbon group, for example, norbornyl, adamantyl, bicyclo[2.2.2]octyl, bicyclo[2.2.1 ]heptyl, bicyclo[3.2.1 ]octyl, or decahydronaphthyl. Alkyl groups optionally can be substituted, for example, with hydroxy (OH), halo, amino, and sulfonyl. An "alkoxy" group is an alkyl group having an oxygen substituent, e.g., --O-alkyl.

The term "alkenyl" refers to straight chained and branched hydrocarbon groups containing carbon atoms having at least one carbon-carbon double bond. Unless otherwise indicated, the hydrocarbon group can contain up to 20 carbon atoms. Alkenyl groups can optionally be substituted, for example, with hydroxy (OH), halo, amino, and sulfonyl.

As used herein, the term "alkylene" refers to an alkyl group having a further defined substituent. For example, the term "alkylenearyl" refers to an alkyl group substituted with an aryl group, and "alkyleneamino" refers to an alkyl groups substituted with an amino group. The amino group of the alkyleneamino can be further substituted with, e.g., an alkyl group, an alkylenearyl group, an aryl group, or combinations thereof. The term "alkenylene" refers to an alkenyl group having a further defined substituent.

As used herein, the term "aryl" refers to a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group, e.g., phenyl or naphthyl. Unless otherwise indicated, an aryl group can be unsubstituted or substituted with one or more, and in particular one to four groups independently selected from, for example, halo, alkyl, alkenyl, OCF.sub.3, NO. sub.2, CN, NC, OH, alkoxy, amino, CO.sub.2H, CO.sub.2alkyl, aryl, and heteroaryl. Exemplary aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, chlorophenyl, methylphenyl, methoxyphenyl, trifluoromethylphenyl, nitrophenyl, 2,4-methoxychlorophenyl, and the like. An "aryloxy" group is an aryl group having an oxygen substituent, e.g., --O-aryl.

As used herein, the term "acyl" refers to a carbonyl group, e.g., C(O). The acyl group is further substituted with, for example, hydrogen, an alkyl, an alkenyl, an aryl, an alkenylaryl, an alkoxy, or an amino group. Specific examples of acyl groups include, but are not limited to, alkoxycarbonyl (e.g., C(O)--Oalkyl); aryloxycarbonyl (e.g., C(O)- -Oaryl); alkylenearyloxycarbonyl (e.g., C(O)--Oalkylenearyl); carbamoyl (e.g., C(O)-- NH.sub.2); alkylcarbamoyl (e.g., C(0)--NH(aikyl)) or dialkylcarbamoyl (e.g., C(O)- NH(alkyl).sub.2).

As used herein, the term "amino" refers to a nitrogen containing substituent, which can have zero, one, or two alkyl, alkenyl, aryl, alkylenearyl, or acyl substituents. An amino group having zero substituents is --NH.sub.2.

As used herein, the term "halo" or "halogen" refers to fluoride, bromide, iodide, or chloride.

As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1 -19 (1977). The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid or inorganic acid. Examples of pharmaceutically acceptable nontoxic acid addition salts include, but are not limited to, salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid lactobionic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p- toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.

Examples

The following Examples have been included to provide illustrations of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications and alterations can be employed without departing from the spirit and scope of the presently disclosed subject matter.

Example 1 . Computational Modeling, Pharmacophore Generation, Virtual and Functional Screening.

In order to generate a productive pharmacophore, computational docking of the prospective METTL3/METTL14/WTAP complex activating compounds was carried out using the complex crystal structures. The structure of the METTL3/METTL14 complex with the S-adenosyl-L-homocysteine (SAFI) was chosen as describing the potential target binding site for a small-molecule inhibitor. The crystal structure of this complex (pdb:5K7W) had been measured by X-ray diffraction with resolution 1 .65 A (Wang P et al., 2016)[30] . The raw crystal structures were corrected and hydrogen atoms were automatically added to the protein using Schrodinger’s Protein Preparation Wizard of Maestro 10.7 (Sastry et al., 2013)[22].

AutoDock Vina 1 .1 .2 (Trott et al., 2010)[29] was used for the docking studies to find out binding modes and binding energies of ligands to the receptor. The number of rotatable bonds of ligand was set by default by AutoDock Tools 1 .5.6 (Morris et al., 2009)[18]. Flowever, if the number was greater than 6, then some of rotatable bonds were made as non-rotatable, otherwise calculations can be inaccurate. The active site, was surrounded with a grid-box sized 65 ^c 65 ^c 65 points with spacing of 1 .0 A. The AutoDock 4.2 force field (Morris et al., 2009)[18] was used in all molecular docking simulations.

The structure of ligand molecules was optimized using the density functional theory B3LYP method (Stephens et al., 1994)[25] with 6-31 G basis set. The molecular dynamics simulations were carried out using Desmond simulation package of Schrodinger LLC (Bowers et al., 2006)[2] The NPT ensemble with the temperature 300 K and pressure 1 bar was applied in all runs. The simulation lengths were 10 ns and 50 ns with relaxation time 1 ps. The OPLS_2005 force field parameters were used in all simulations (Banks et al., 2005)[1 ]. The long range electrostatic interactions were calculated using the Particle Mesh Ewald method (Toukmaji et al., 1996)[26]. The cutoff radius in Coloumb^' interactions was 9.0 A. The water molecules were described using SPC (simple point charge) model (Zielkiewicz et al., 2006)[33]. The behavior and interactions between the ligands and enzyme were analyzed using the Simulation Interaction Diagram tool implemented in Desmond molecular dynamics package. The stability of molecular dynamics simulations was monitored by looking on the root mean square deviation (RMSD) of the ligand and protein atom positions in time.

As reported by Wang P et a/.[30], there are several distinct regions of probable interactions between the ligand and enzyme. As confirmed by our molecular docking calculations, the amino group of the adenosyl fragment of SAH is hydrogen bonded with Asp377 of the Mettl3 (cf. Figure 3). The binding is further supported by another bond between the adenine N1 atom and an adjacent peptide bond NH group. The adenine ring is sandwiched between Phe534 and Asn549, while many polar contacts help to hold the hydroxyl groups on the ribose as well as the amino and carboxyl groups of SAH. The terminal amino group of SAH is acting as hydrogen bond donor to the Asp395 of the catalytic center of enzyme.

Based on this structure we proceeded with the search of effectively bound small molecule fragments. The first group of fragments consisted of various substituted purines. In variance to the crystal structure of SAH itself, these compounds tend to be bound to the region of the Mettl3 protein involving Asp395, Phe534, Arg536 and Asn539 (Figure 4).

A virtual screening on ZINC (Irwin et al., 2005)[27] and DrugBank 4.0 (Law et al., 2014)[28] databases was carried out using nitrogen-containing heterocycles as base structures. Remarkably, we found a series of fragments with piperidine and piperazine rings having exceptionally high docking efficiencies. The docking free energies and docking efficiencies of the best fragment compounds are given in Table 1 . The molecular dynamics simulations were thereafter carried out for two compounds, representing the two different promising scaffolds, the compounds (IV) and (VI), respectively.

The modeling results explain the mechanism of the activation of METTL3/METTL14 by the studied compounds. The docking and molecular dynamics simulations of active compounds show that the piperidine and piperazine rings of these small ligands are deeply embedded into the structure of METTL3/METTL14 protein. The simultaneous docking of compound (IV) and SAM to the protein displays the close proximity of these two compounds in the active centre of the protein (Figure 5). The interaction between the carbonyl oxygen atoms of the studied series of ligands and the methylation reaction centre at the sulphur atom of the methionine group of SAM increases the binding affinity of the latter and lower the energy barrier of the substrate RNA methylation reaction, thus making these compounds effective RNA methylation activators.

Example 2. Methods

Cell lines. HEK-293 (ATCC) cells were cultured in DMEM (Gibco), supplemented with 10% FBS (Gibco) and penicillin-streptomycin (Gibco) at 37°C and 5% CO2.

Plasmids. The plasmids for Mettl3/14 protein complex production: pcDNA3/Flag- METTL3 (Addgene plasmid # 53739) and pcDNA3/Flag-METTL14 (Addgene plasmid # 53740) (Liu et al„ 2014)[16]

Proteins. HEK-293 cells were transiently co-transfected with 25 pg of each Mettl3 and MettH 4 plasmids using Lipofectamine® 2000 (Invitrogen). Isolation of the METTL3/METTL14 complex comprised of lysis of HEK-293 cells 48h post-transfection and purification of the lysate with the ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich) (cf. Supplementary Figure 1 ). The complex was eluted with 150 ng/ml 3x FLAG® peptide (Sigma-Aldrich).The anti-flag purified proteins were denaturated and run on Mini- PROTEAN precast 4-20% gels (Bio-Rad), 1 pg per well. Precision Plus Protein Dual Color Standard from Bio-Rad was used as a ladder. The proteins were transferred onto an Immunobilon FL PVDF membrane (Merck Millipore) and blocked with the blocking buffer in PBS (Licor). The membrane probed for M-a-FLAG was blocked in 5% non-fat milk in TBST (20mM Tris, 150mM NaCI, pH 7.4, 0,1 % Tween 20), the same solution was used for M-a-FLAG (1 :1000) primary antibody dilution. Rb-a-Mettl3 (1 :2000), Rb- a-Mettl14 (1 :200) and M-a-WTAP (1 :200) were diluted in blocking buffer (Licor). Blocking was done at RT on shaker for 1 h and primary antibody incubations at +4C° on shaker O/N. The membranes were washed 3 times with PBST (137mM NaCI, 2.7mM KCI, 4.3mM Na2HP04, 1 .47mM KH2P04, 0,1 % Tween 20), and placed into the secondary antibody solution. In case of anti-Flag antibody all the washes were done with TBST. Secondary antibodies Gt-a-Rb IRDye 800CW and Gt-a-M IRDye 680LT were diluted in blocking buffer (Licor) 1 :10000. Membranes were incubated with secondary antibody for 1 h at RT in the dark. After subsequent washes, the membranes were imaged using Odyssey CLx (Licor).

Example 3. Surface plasmon resonance measurements of ligand-protein binding

SPR measurements were performed with a Biacore T100 (GE Healthcare Life Sciences) at 25^°C. The instrument was cleaned using an in-build“desorb” protocol before a new CM5 series S sensor chip (GE Healthcare Bio-Sciences) was docked and primed at least three times with a 1 .02x PBS-P+ buffer (10x PBS-P+, GE Healthcare Bio-Sciences). After the priming, the sensor chip was preconditioned with two 20 pi injections, each of 50 mM NaOH, 100 mM HCI and 0.05% SDS with a flow rate of 100 mI/min. After preconditioning, the detector signal was normalized with 70% glycerol (GE Healthcare Bio-Sciences) and the system was re-primed. All used glassware was rinsed with 50 mM NaOH and filtered, deionized water before use and all used buffers were sterile filtered and degassed before each experiment. a2-Macroglobulin (10 pg/ml) and METTL3/METTL14 (100 pg/ml) diluted in 10 mM sodium acetate (GE Healthcare Bio-Sciences) with and without 50 mM NaCI (pH 4.0), were immobilized on reference and active flow cells, respectively, to a surface density of approximately 12000 RU by using standard amine coupling (Johnsson et al ., 1991 ). The carboxymethyl dextran surface was activated with a 7-min injection of a 1 :1 ratio of 0.4 M EDC ((1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide, GE Healthcare Bio- Sciences) and 0.1 M NHS (N-hydroxysuccinimide, GE Healthcare Bio-Sciences). Immobilized proteins were injected onto the flow cells using a flow rate of 10 mI/min for 420 s and remaining amine-reactive NHS-esters were blocked with 1 M ethanolamine- HCI (pH 8.0, GE Healthcare Bio-Sciences) using 1 02x PBS-P+ as a running buffer.

All tested compounds (compounds (III), (IV), (V) and (VI)) demonstrated METTL3/METTL14/WTAP binding in a concentration-dependent manner. Smoothed ligand association-dissociation curves and the compounds’ dissociation constants KD are shown in Table 2. The effect of compounds (IV) and (VI) at variable concentrations on SAM binding to the METTL3/14/WTAP enzyme. A constant concentration of the compounds (IV) (1 nM to 100 nM) or (VI) (25 DM) in the running buffer was used in separate Biacore experiments to determine their effects on the KD for SAM binding (Table 3). The results demonstrate that suggesting that the studied small-molecule ligands act by enhancing the binding of SAM by several orders of magnitude.

Example 4. METTL3/METTL14/WTAP enzymatic assay activity of small molecules on

RNA mrethylation

The enzymatic assay was modified from Li et al. (Li et al., 2016)[12]. The experiments were conducted in reaction buffer (20mM Tris pH7.5, 1 mM DTT, 0,01 % Triton™ X- 100, 40U/100mI buffer RNaseOUT™ (Invitrogen)). The reaction mixture contained 200nM unmethylated N6-adenine single-stranded-RNA probe with a biotin tag (5’- uacacucgaucuggacuaaagcugcuc-biotin-3’, Integrated DNA Technologies), 500nM tritiated S-(5'-adenosyl)-L-methionine (3H-SAM, Perkin Elmer) and 5mM purified METTL3/14 complex. DMSO content, as a solvent for small molecules in the enzymatic reaction, constituted 0,1 %. Enzymatic assay reactions were incubated for 20h at 21 °C on shaker, transferred to wells on streptavid in-coated 96-well plate (Perkin Elmer) and incubated for additional 1 h at room temperature. After that, the plate was washed with sterile 20mM Tris pH7.5 2x, the results were acquired using 2450 MicroBeta® liquid scintillation counter (Wallack). The scintillation counts were proportional to amount of methylated RNA.

The methylation activity of METTL3/METTL14/WTAP complex was measured by the amount of tritiated substrate (3H-S-adenosylmethionine, SAM) in the samples treated by the small compounds (III) - (VI). A specific METTL3/METTL14/WTAP-methylatable RNA oligonucleotide sequence 5’-uacacucgaucuggacuaaagcugcuc-biotin-3’was utilized in a radioactivity-based assay to evaluate the effect of the designed ligands on the activity of HEK-293 cell-expressed and FLAG-tag-purified METTL3/METTL14/WTAP (Figure 7). As controls for the assay, the effects of the non- tritiated SAM substrate and its demethylated analogue SAH were evaluated. Both non- tritiated SAM and SAH inhibited METTL3/METTL14/WTAP-mediated enzymatic 3H- methyl label incorporation to the specific substrate oligonucleotide probe.

The results indicate the tested compounds increase significantly the METTL3/METTL14/WTAP complex activity. As evaluated from the enzymatic assay results, the EC50 values for all tested compounds are given in Figure 7. In terms of their increasing EC50s the order of the activating compounds were (III) < (VI) < (V) < (IV). The compound (III) showed the most potent METTL3/METTL14/WTAP enzyme activating effect (cf. Figure 9(c)). Therefore, the compounds activate RNA methylation and can be therefore act as therapeutic agency in the case of pathologies involving deficiency of RNA m6A methylation.

Table 1. The compounds with the highest docking efficiencies to METTL3/METTL14 complex.

Table 2. Surface plasmon resonance measurements of the interactions between METTL3 and small-molecule ligands. Kd - binding constant (M), k_a - association rate constant (M ¹s ¹), kd - dissociation rate constant (s ¹).

Table 3. Surface plasmon resonance measurements of the interactions between METTL3 and SAM in the presence of the small-molecule ligands. Kd - binding constant (M), k_a - association rate constant (M^_1s ¹), kd - dissociation rate constant (s ¹).

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Claims

1 . A method of modulating the RNA methylation at 6-position of adenine (m6A) by effective amount of a compound having binding and/or activation for a METTL3/METTL14 methyltransferase complex.

2. The method of claim 1 , wherein a compound has binding and/or activation for a METTL3/METTL14/WTAP complex.

3. The method of claim 1 , wherein the compound is a small molecule.

4. The method of claim 1 , when it is combined with the inhibitors of RNA demethylases FTO and AlkBH5.

5. The method of claim 3, wherein the compound has a structure of Formula (I)

wherein: R1 and R2 are independently selected from the group consisting of H, alkyl, aryl, aralkyl, acyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, alkyl carbamoyl, and dialkylcarbamoyl, aminoalkyl, aminoalaryl; or a pharmaceutically acceptable salt thereof.

6. The method of claim 3, wherein the compound has a structure of Formula (II)

wherein: R1 and R2 are independently selected from the group consisting of H, alkyl, aryl, aralkyl, acyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, alkylcarbamoyl, and dialkylcarbamoyl, aminoalkyl, aminoalaryl; or a pharmaceutically acceptable salt thereof.

7. The method of claim 3, wherein the compound has a structure of Formula (III)

8. The method of claim 3, wherein the compound has a structure of Formula (IV)

9. The method of claim 3, wherein the compound has a structure of Formula (V)

0. The method of claim 3, wherein the compound has a structure of Formula (VI)