Method of survival and protection of neurons by inhibitors of RNA m6A demethylases FTO and ALKBH5

Abstract

A method of support of dopaminergic neuron survival using an RNA methylation modulator compound by inhibiting an RNA m6A demethylase, wherein the RNA m6A demethylase is fat mass and obesity-associated

Description

Inventors: Mart Saarma, Li-Ying Yu, Neinar Seii, Simona Selberg, Mati Karelson Assignee: Chemestmed, Ltd. Technical Field The presently disclosed subject matter generally relates to the epitrancriptomic regulation of ribonucleic acid (RNA) methylation through the use of small-molecule inhibitors of the RNA m6A demethylases FTO and AlkBH5, and their application to support the neuronal cell survival and neuroprotection. Background Art It has been shown that the RNA m6A modification is important in brain development, neuron signalling and neuronal disorders (Angelova, et al, 2018; Jung, et al, 2018; Noack, et al, 2019; Widagdo, et al, 2018; Du, et al, 2019; Livneh, et al, 2020). For instance, it has been demonstrated that m6A-dependent mRNA decay is critical for proper transcriptional prepatteming in mammalian cortical neurogenesis (Yoon, et al, 2017). Weng et al. had uncovered an epitranscriptomic mechanism wherein axonal injury elevates m6A levels and signaling to promote protein translation, including regeneration-associated genes, which is essential for functional axon regeneration of peripheral sensory neurons (Weng, et al, 2018). It has been shown on the limited patient group (Chinese Han population) that m6A genes may play a role in conferring risk of dementia (Du, et al, 2015). Different m6A regulating enzymes are involved directly in the processes of learning and memory. Through its binding protein YTHDF1, m6A promotes protein translation of target transcripts in response to neuronal stimuli in the adult mouse hippocampus, thereby facilitating learning and memory (Shi, et al, 2018). Similarly, the RNA m6A demethylase FTO is expressed in adult neural stem cells and neurons and displays dynamic expression during postnatal neurodevelopment. It has been demonstrated that the deficiency of FTO could reduce the proliferation and neuronal differentiation of adult neural stem cells in vivo, leading to impaired learning and memory (Li, et al, 2017). In addition, the inactivation of the Fto gene, encoding a nucleic acid demethylase, impairs dopamine receptor type 2 (D2R) and type 3 (D3R)-dependent control of neuronal activity and behavioural responses (Hess, et al, 2013). The m6A homeostasis in neurons are controlled by more than one regulator. Thus, it was found that global m6A modification of mRNAs is down-regulated in 6-OHDA-induced rat pheochromocytoma PC12 cells originating from adrenal medulla and the striatum of 6-OHDA treated rat brain that is the animal model of Parkinson’s disease (PD). The reduction of the m6A level in PC12 cells by. The reduction of the m6A level in PC 12 cells by overexpressing a nucleic acid demethylase, FTO, or by m6A inhibitor cycloleucine could induce the expression of N-m ethyl-D-aspartate (NMDA) receptor 1, and elevate oxidative stress and Ca2+ influx, resulting in PC12 neuron apoptosis (Chen, et al, 2019). Based on above, the dysregulation of the m6A can possibly be related to neurodegeneration in Parkinson’s disease. In summary, either the compensatory upregulation or downregulation of m6A could be needed in neuronal cells, depending on their physiological or pathological state. It has been shown (Chen, et al, 2019) that the downregulation of m6A is involved in dopamine neuronal death. Therefore, the inhibitors of the RNA m6A demethylases FTO or AlkBH5 should support the homeostasis of m6A in dopamine neurons, support their survival in stress conditions and protect them from neurotoxin-induced cell death. Summary of Invention The present invention is related to a method of supporting the survival of the neuronal cells and protecting them from cell death induced by neurotoxins or injury using the inhibitors of the RNA m6A demethylases FTO and AlkBH5. Also disclosed are the compounds, or salts or esters thereof, which can protect and support the survival of the neuronal cells. The compounds described can be used for treating Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer’s disease, drug addiction, traumatic brain injury, psychosis, depression, multiple sclerosis and nausea by administering to the subject one or more compounds, or a pharmaceutically acceptable salt thereof. 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 Figure 1. The binding site of the compound (V) (FTO inhibitor) to FTO protein. Figure 2. The inhibitory effect IE of the compound (V) on the demethylation of the probe RNA by FTO. Figure 3. The inhibitory effect IE of the compound (VI) on the demethylation of the probe RNA by FTO. Figure 4. The effect of the FTO inhibitors (V) and (VI) on the survival of the dopaminergic neurons. Figure 5. The effect of the AlkBH5 inhibitors (VII) and (VIII) on the survival of the dopaminergic neurons. Detailed description of Invention Disclosed herein are compounds and methods of supporting the survival of the neuronal cells and protecting them from neurotoxin- or injury-induced cell death by using the RNA m6A methyltransferase complex METTL3 / METTL14 / WTAP activators. 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, the compound supporting the survival of neuronal cells has a structure of Formula (I), wherein: RI is amino-group or halogen; and R2 is selected from the group consisting of H, alkyl, aryl, or aralkyl,; or a pharmaceutically acceptable salt thereof. In some embodiments, the compound supporting the survival of neuronal cells has a structure of Formula (II) wherein: RI and R2 are independently, hydrogen, amino-group or halogen;; and R3 is selected from the group consisting of H, alkyl, aryl, or aralkyl,; or a pharmaceutically acceptable salt thereof. In some embodiments, the compound supporting the survival of neuronal cells has a structure of Formula (III) wherein: RI and R2 are independently selected from the group consisting of H, alkyl, aryl, aralkyl, acyl, alkoxy carbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, alkylcarbamoyl, and dialkylcarbamoyl, aminoalkyl, aminoalaryl; or a pharmaceutically acceptable salt thereof. In some embodiments, the compound supporting the survival of neuronal cells has a structure of Formula (IV) (IV) wherein: RI is selected from the group consisting of H, alkyl, aryl, aralkyl, acyl, alkoxy carbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, alkylcarbamoyl, and dialkylcarbamoyl, aminoalkyl, aminoalaryl; or a pharmaceutically acceptable salt thereof. In some embodiments, the compound supporting the survival of neuronal cells has a structure of Formula (V) or a pharmaceutically acceptable salt thereof. 5 In some embodiments, the compound supporting the survival of neuronal cells has a structure of Formula (VI) ci (VI) or a pharmaceutically acceptable salt thereof In some embodiments, the compound supporting the survival of neuronal cells has a structure 10 of F ormul a (VII) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound supporting the survival of neuronal cells has a structure of Formula (VIII) 15 or a pharmaceutically acceptable salt thereof. The compounds described can be used for treating Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer’s disease, drug addiction, traumatic brain injury, psychosis, depression, multiple sclerosis and nausea by administering to the subject one or more compounds, 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.l6 bicyclic or polycyclic hydrocarbon group, for example, norbomyl, 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 (eg., 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(O)-NH(alkyl)) 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, dodecyl sulfate, 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. High-throughput virtual screening of compound libraries for the identification of potential inhibitors of the RNA m6A demethyl ase FTO. The virtual screening of the compound libraries requires the availability of the 3D structure of the target protein, in this case FTO. The crystal structure of the RNA demethylase FTO has been published as for the pure protein (Han, et al, 2010) and together with different ligands (Aik, et al, 2013; Huang, et al, 2014; Toh, et al, 2015). Crystal structure of FTO in complex with 5-carboxy-8-hydroxyquinoline (pdb: 4IE4) was chosen for further molecular docking modeling. The structure had been determined using X-ray diffraction with the resolution 2.5 A. (Aik, et al, 2013). The choice of this FTO structure was made on the basis of the availability of the ligand compounds. The raw crystal structure was corrected and hydrogen atoms were automatically added to the protein using Schrodinger’s Protein Preparation Wizard of Maestro 10.7 (Sastry, et al, 2013). AutoDock 4.2 (Morris et al., 2009) 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). However, 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 80 x 80 x 80 points with spacing of 0.375 A” The AutoDock 4.2 force field was used in all molecular docking simulations. The docking efficiencies (DE) were calculated as follows DE= (AG_dock) / N (1) where AGdock is the docking free energy and N - the number of non-hydrogen (“heavy”) atoms in the ligand molecule. The structure of ligand molecules was optimized using the density functional theory B3LYP method with 6-31G basis set (Stephens, et al, 1994). The virtual screening was carried out on ZINC database (Irwin, et al, 2005). The structures and binding free energies of the compounds with the highest docking efficiencies to FTO protein are given in Table 1. The structures of the binding complex of the compound (V) in the FTO active centre and is presented on Figure 1. Example 2. Screening of computationally predicted RNA m6A demethylase FTO ligands in enzyme inhibition assay. The enzymatic assay was modified from Huang et al. 2015 (Huang et al., 2015). The experiments were conducted in reaction buffer (50 mM Tris-HCl, pH 7.5, 300 pM 2OG, 280 pM (NH4)2Fe(SO4)2 and 2 mM L-ascorbic acid). The reaction mixture contained 200 ng methylated N6-adenine RNA probe (SEQ ID NO: 1) (5’-CUUGUCAm6ACAGCAGA-3’, Dharmacon) and lOnM FTO protein. Reactions were incubated on 96-well plate for 2h at RT. After that, the amount of m6A that was measured using EpiQuik m6A RNA methylation Quantification Colorimetric Kit (Epigentek). The inhibitory effect IE of compounds on RNA probe demethylation by FTO was calculated as the enhancement of the m6A amount as compared to the negative control (DMSO) relative to the difference between m6A amounts of the positive control (max inhibition) and the negative control (eq. (2)): =.----. ----- (2) where Cinh, Cmh(max) and CDMso are the amounts of m6A at a given concentration of the inhibitor, maximum inhibition and in the case of DMSO, respectively. The dependence of the IE on the inhibitor concentration for the compound (V) is shown on Fig. 2 and for the compound (VI) on Fig. 3. The inhibitory concentrations are IC50 = 28.9 DM for the compound (V) and IC50 = 1.46 DM for the compound (VI), respectively. Therefore, both compounds are efficient inhibitors of the RNA m6A demethylase FTO. Example 3. Treatment of primary cultures of midbrain dopaminergic neurons by m6A inhibitors of RNA m6A demethyl ases FTO and AlkBH5. The midbrain floors were dissected from the ventral mesencephalic of 13 days old NMRI strain mouse embryos. The tissues were incubated with 0.5% trypsin (103139, MP Biomedical) in HBSS (Ca2+ / Mg2+-free) (14170112, Invitrogen) for 20 min at 37°C, then mechanically dissociated. Cells were plated onto the 96-well plates coated with poly-L-ornithine (Sigma-Aldrich). Equal volumes of cell suspension were plated onto the center of the dish. The cells were grown for 5 days with different concentrations of FTO or AlkBH5 inhibitor compounds separately. Human recombinant GDNF (100 ng / ml) (Icosagen) or a medium without any neurotrophic compound added were used as positive and negative controls, respectively. After growing 5 days, the neuronal cultures were fixed and stained with anti-Tyrosine Hydroxylase antibody (MAB318, Millipore Bioscience Research Reagents. Images were 5 acquired by Cellinsight high-content imaging equipment. Immunopositive neurons were counted by CellProfiler software and the data were analyzed using CellProfiler analyst software. (Yu, et al, 2008; Mahato et al, 2020). The measurements were carried out with the FTO inhibitors (compounds (V) and (VI)) and AlkBH5 inhibitors (compounds (VII) and (VIII)) (Selberg, et al, 2019). The results are expressed as % of cell survival compared to 10 GDNF-maintained neurons (Figures 4 and 5). It can be seen that the compounds have some effect already at the 10 nM concentration and comparable to the neurotrophic factor GDNF effect at their 1 000 nM concentration. Therefore, these compounds are efficiently supporting the survival of neurons at growth factor deprivation-induced stress conditions. Table 1. The compounds with the highest docking efficiencies to FTO protein. Compound structure □ G (kcal / mol) DE OO ‘ -7.37 0.53 (VI) -7.70 0.51 T* (v) -7.03 0.50 References Cited Aik, W.S.; Demetriades, M.; Hamdan, M.K.K.; Bagg, E.A.L.; Yeoh, K.K.; Lejeune, C.; Zhang, Z.; McDonough, M.A.; Schofield, C.J. (2013). Structural basis for Inhibition of the Fat Mass and Obesity Associated Protein (FTO). J. Med. Chem. 56, 3680-3688. Aik, W.; Scotti, J.S.; Choi, H.; Gong, L.; Demetriades, M.; Schofield, C.J.; McDonough, M.A. (2014)Structure of human RNA N6-methyladenine demethylase ALKBH5 provides insights into its mechanisms of nucleic acid recognition and demethylation. Nucleic Acids Res. 42, 4741-54. Angelova M.T, Dimitrova D.G, Dinges N, Lence T, Worpenberg L, Carre C, Roignant J.Y. (2018). The Emerging Field of Epitranscriptomics in Neurodev el opmental and Neuronal Disorders. Front. Bioeng. Biotechnol. 6, 46. Chen X, Yu C, Guo M, Zheng X, Ali S, Huang H, Zhang L, Wang S, Huang Y, Qie S, Wang J. 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Claims

1. Compound that modulates RNA methylation by inhibiting a RNA m6A demethylase, for use in the treatment of a disease selected from the group consisting of Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, drug addiction, traumatic brain injury, psychosis, depression, multiple sclerosis and nausea, wherein the RNA m6A demethylase is a fat mass and obesity-associated protein (FTO), and wherein the compound is selected from the group consisting of:(a) a small molecule having the structure of Formula (I):18 07 25wherein: Ri is amino-group or halogen; and R2 is selected from the group consisting of H, alkyl, aryl, or aralkyl; or a pharmaceutically acceptable salt thereof; or(b) a compound having the structure of Formula (VI):(VI)or a pharmaceutically acceptable salt thereof.

2. Compound that modulates RNA methylation by inhibiting a RNA m6A demethylase, for use in supporting the survival of dopaminergic neurons, wherein the RNA m6A demethylase is a fat mass and obesity-associated protein (FTO), and wherein the compound is selected from the group consisting of:(a) a small molecule having the structure of Formula (I):wherein: Ri is amino-group or halogen; and R2 is selected from the group consisting of H, alkyl, aryl, or aralkyl; or a pharmaceutically acceptable salt thereof; or(b) a compound having the structure of Formula (VI):18 07 25(VI)or a pharmaceutically acceptable salt thereof.

3. Compound for use according to claim 2, wherein the supporting the survival of dopaminergic neurons is protecting the dopaminergic neurons from cell death induced by neurotoxins or injury.

4. Compound for use according to any one of claims 1 to 3, wherein the compound is a small molecule having the structure of Formula (I):wherein: Ri is amino-group or halogen; and R2 is selected from the group consisting of H, alkyl, aryl, or aralkyl; or a pharmaceutically acceptable salt thereof.

5. Compound for use according to claim 4, wherein the compound has the structure of Formula (V):or a pharmaceutically acceptable salt thereof.

6. Compound for use according to any of claims 1 to 3, wherein the compound has the structure of Formula (VI):COor a pharmaceutically acceptable salt thereof.

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