WO2019197024A1 - Procédé de modulation de la méthylation de l'arn - Google Patents

Procédé de modulation de la méthylation de l'arn Download PDF

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WO2019197024A1
WO2019197024A1 PCT/EP2018/059284 EP2018059284W WO2019197024A1 WO 2019197024 A1 WO2019197024 A1 WO 2019197024A1 EP 2018059284 W EP2018059284 W EP 2018059284W WO 2019197024 A1 WO2019197024 A1 WO 2019197024A1
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mettl3
rna
compound
mettl14
wtap
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PCT/EP2018/059284
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English (en)
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Simona SELBERG
Daria BLOKHINA
Esko Kankuri
Mati Karelson
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Chemestmed Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase

Definitions

  • RNA ribonucleic acid
  • RNA stability 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 ] .
  • 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.
  • RNA methyltransferase enzyme complex METTL3/METTL14/WTAP consisting of three components: METTL3
  • RNA demethylases FTO fat mass and obesity-associated protein
  • AlkBH5 AlkB family member 5
  • 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.
  • 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].
  • 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 ].
  • 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.
  • 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.
  • the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above.
  • 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.
  • various aspects and features of the invention can be combined, creating additional aspects which are intended to be within the scope of the invention.
  • 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
  • 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).
  • the compound is administered in a composition that also includes one or more pharmaceutically acceptable diluents, adjuvants, or carriers.
  • METTL3/METTL14/WTAP complex activator has a structure of Formula (I),
  • 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.
  • the METTL3/METT14/WTAP complex activator compound has a structure of Formula (II)
  • 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.
  • the METTL3/METTL14/WTAP complex activator compound has a structure of Formula (III)
  • the METTL3/METTL14/WTAP complex activator compound has a structure of Formula (IV)
  • the METTL3/METTL14/WTAP complex activator compound has a structure of Formula (V)
  • the METTL3/METTL14/WTAP complex activator compound has a structure of Formula (VI)
  • 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.
  • 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.
  • alkylene refers to an alkyl group having a further defined substituent.
  • alkylenearyl refers to an alkyl group substituted with an aryl group
  • 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.
  • alkenylene refers to an alkenyl group having a further defined substituent.
  • 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.
  • 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.
  • 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.
  • 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).
  • alkoxycarbonyl e.g., C(O)--Oalkyl
  • aryloxycarbonyl e.g., C(O)- -Oaryl
  • alkylenearyloxycarbonyl e.g., C(O)--Oalkylenearyl
  • carbamoyl
  • 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.
  • halo or halogen refers to fluoride, bromide, iodide, or chloride.
  • 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.
  • 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.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • 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.
  • 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, pam
  • 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.
  • Example 1 Computational Modeling, Pharmacophore Generation, Virtual and Functional Screening.
  • 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 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.
  • 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 tension 2014)[16]
  • 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).
  • 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.
  • EDC ((1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide, GE Healthcare Bio- Sciences)
  • NHS N-hydroxysuccinimide, GE Healthcare Bio-Sciences
  • Immobilized proteins were injected onto the flow cells using a flow rate of 10 mI
  • 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 % TritonTM X- 100, 40U/100mI buffer RNaseOUTTM (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.
  • 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).
  • m6A RNA Methylation Regulates the Self-Renewal and Tumorigenesis of Glioblastoma Stem Cells. Cell Rep. 18, 2622- 2634.
  • MiR-33a suppresses proliferation of NSCLC cells via targeting METTL3 mRNA. Biochem. Biophys. Res. Commun. 482, 582-589. 7.
  • N6-Methyl- Adenosine (m6A) in RNA An Old Modification with A Novel Epigenetic Function.
  • DrugBank 4.0 Shedding New Light on Drug Metabolism. Nucleic Acids Res. 42, D1091 -D1097. 29. Trott, O.; Olson, A. J. (2010). AutoDock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization and Multithreading. J. Comput. Chem. 31 , 455-461.
  • Reduced m6A mRNA methylation is correlated with the progression of human cervical cancer, Oncotarget. 8, 98918-98930.
  • Promoter-bound METTL3 maintains myeloid leukaemia by m6A-dependent translation control. Nature 552, 126-131.

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Abstract

L'invention concerne des procédés et des composés qui modulent spécifiquement la méthylation de l'ARN par activation du complexe ARN méthyltransférase METTL3/METTL14. Dans certains modes de réalisation, le composé se lie à et/ou active un complexe METTL3/METTL14/WTAP.
PCT/EP2018/059284 2018-04-11 2018-04-11 Procédé de modulation de la méthylation de l'arn WO2019197024A1 (fr)

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WO2021081211A1 (fr) * 2019-10-25 2021-04-29 Accent Therapeutics, Inc. Modulateurs de mettl3
WO2021111124A1 (fr) * 2019-12-02 2021-06-10 Storm Therapeutics Limited Composés polyhétérocycliques en tant qu'inhibiteurs de mettl3
WO2021116476A1 (fr) * 2019-12-12 2021-06-17 Chemestmed Ltd. Agonistes de la méthylation de l'adénosine n -6 d'arn pour une utilisation dans la suppression de l'activation et de la réplication du provirus latent du vih-1
WO2022013420A1 (fr) * 2020-07-17 2022-01-20 European Molecular Biology Laboratory Biomarqueurs pronostiques pour le cancer
WO2022074391A1 (fr) * 2020-10-08 2022-04-14 Storm Therapeutics Limited Composés inhibiteurs de mettl3
GB2601519A (en) * 2020-12-02 2022-06-08 Chemestmed Ltd Method of support of survival and protection of neurons by RNA m6A methyltransferase complex METTL3/METTL14 activators
WO2022243333A1 (fr) * 2021-05-17 2022-11-24 Universität Zürich Inhibiteurs de n6-adénosine-méthyltransférase dans le traitement du cancer

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