WO2020114583A1

WO2020114583A1 - A method of modulating hiv-1 provirus activation and replication

Info

Publication number: WO2020114583A1
Application number: PCT/EP2018/083549
Authority: WO
Inventors: Simona SELBERG; Eva ŽUSINAITE; Andre MERITS; Mati Karelson
Original assignee: Chemestmed Ltd.
Priority date: 2018-12-04
Filing date: 2018-12-04
Publication date: 2020-06-11

Abstract

Methods and compounds are reported that specifically modulate HIV-1 provirus activation and replication of emerged virus genome 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 HIV-1 proVIRUS ACTIVATION AND REPLICATION Technical Field

The presently disclosed subject matter generally relates to the activation of the latent HIV-1 provirus using the epitranscriptomic regulation of the ribonucleic acid (RNA) methylation.

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 (Helm et al., 2017). The most abundant modification in eukaryotic messenger RNA is N6-methyladenosine (m6A) (Roundtree et al., 2017). 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). The m6A presence in RNA is regulated by specific enzymes, i.e. the RNA methyltransferases, RNA methylases and RNA reader proteins.

The modification of the viral RNAs by methylation of the amino-group at 6-position of adenosine (m6A) has been known for some time (Beemon et al, 1977; Finkel et al, 1983). It has been shown that the presence of m6A is regulating the HIV-1 RNA viral replication and gene expression (Kennedy et al., 2016; Lichinchi et al., 2016, Tirumuru, et al, 2016; Rana, 2018).

The methylation of the adenosine is dynamically regulated in mammalian cells by RNA methyltransferases or “writers”, demethylases or “erasers” and m6A recognizing proteins or“readers”. The N6-methylation of adenosine is catalyzed by a 200 kDa methyltransferase heterodimer complex consisting of the Methyltransferase-Like Protein 3 (METTL3), METTL14 and the associated proteins Wilms Tumor 1 Associated Protein (WTAP), RBM15/RBM15B and KIAA1429 (Liu et al, 2014; Meyer et al, 2017). METTL3 is a S-adenosylmethionine (SAM) dependent RNA m6A methyltransferase, while METTL14 together with RBM15/RBM15B, plays an important role in substrate recognition and binding (Wang, P, et al, 2016; Wang, X, et al, 2016; Patil et al, 2016). The primary function of WTAP is to localize METTL3 and METTL14 to nuclear speckles. It has been shown that WTAP depletion causes loss of METTL3 and METTL14 localization from these speckles and loss of m6A formation in mRNA (Ping et al. 2014). Thus, WTAP maintains METTL3 in speckles to efficiently methylate mRNA.

Recently, we have reported the discovery of the small-molecule activators of the RNA m6A methyltransferase METTL/METTL14/WTAP complex (Selberg et al, 2018). These activators of the m6A writer complex provide the first upstream means for increasing cellular m6A amounts. Contrary to FTO or AlkBH5 inhibitors that rely on the baseline activity of m6A writing to be effective, these small molecule m6A writer activators can help, for example, targeted guidance of cells to specific phenotypes. Based on the available knowledge on the m6A dynamics in HIV-1 provirus activation and subsequent virus replication as briefly discussed above, the increase of m6A in host cell and/or HIV RNA should enhance the virus replication. Whereas this discards the small-molecule activators of the METTL/METTL14/WTAP complex as possible antivirals, the compounds may be of large interest as activating the latent HIV provirus copies deposited in host cells’ genome (Kim et al, 2018; Marsden et al, 2018). Cells containing activated provirus can be subsequently targeted by use of conventional anti-HIV drugs.

In the current work, we have studied the activity of the small-molecule activators of the RNA m6A methyltransferase METTL/METTL14/WTAP complex on the HIV-1 provirus activation and subsequent virus replication. In parallel, we monitored the dynamics of the m6A as influenced by the activation of the m6A methylation in both HIV-1 and cellular RNA.

Summary of Invention

The present invention is related to a method to increase the HIV-1 provirus activation and subsequent replication (virion formation) through the activation of the RNA m6A methyltransferase METTL3/METTI14/WTAP complex. Also disclosed are the compounds, or salts or esters thereof, which can increase the HIV-1 provirus activation and subsequent replication.

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 HIV-1 virus production measured by p24 ELISA (T, OD450) after treatment with the RNA m6A methyltransferase METTL3/METTL14/WTAP activators at different compound concentrations (a) Compound (VIII); (b) compound (IX); (c) compound (X).

FIG 2. HIV-1 virus production measured by the luciferase activity (LUC) after treatment with the viral assay media pre-treated with METTL3/METTL14/WTAP activators at 1 mM (1 micromolar = 10 ³ mol/m³) concentration.

Detailed description of Invention

Disclosed herein are compounds and methods of increasing the HIV-1 provirus activation and subsequent HIV-1 replication through activation of the RNA m6A methyltransferase 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. Disclosed is a method of stimulating the HIV-1 provirus activation and subsequent replication by using compounds comprising agonists of RNA adenosine N-6 methylation wherein said compounds comprising agonists of RNA adenosine N-6 methylation are activators of the RNA methyltransferase METTL3/METTL14/WTAP complex.

The compound can be a small molecule. In some embodiments, HIV-1 provirus activation and replication increasing 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, alkylcarbamoyl, and dialkylcarbamoyl, aminoalkyl, aminoalaryl; or a pharmaceutically acceptable salt thereof.

In some embodiments, HIV-1 provirus activation and replication increasing 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; or a pharmaceutically acceptable salt thereof. 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, HIV-1 provirus activation and replication increasing compound has a structure of Formula (III)

wherein: R1 , R2, R3 and R4 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, HIV-1 provirus activation and replication increasing compound has a structure of Formula (IV)

In some embodiments, HIV-1 provirus activation and replication increasing compound has a structure of Formula (V) wherein: R1 , R2, R3 and R4 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, HIV-1 provirus activation and replication increasing compound has a structure of Formula (VI)

In some embodiments, HIV-1 provirus activation and replication increasing compound has a structure of Formula (VII)

In some embodiments, HIV-1 provirus activation and replication increasing compound has a structure of Formula (VIII)

or a pharmaceutically acceptable salt thereof.

In some embodiments, HIV-1 provirus activation and replication increasing compound has a structure of Formula (IX)

or a pharmaceutically acceptable salt thereof.

In some embodiments, HIV-1 provirus activation and replication increasing compound has a structure of Formula (X)

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(alkyi)) or dialkylcarbamoyl (e.g., C(0)~ 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.

Compounds.

Methyl 6-methyl piperidine-3-carboxylate (VII) (ArkPharm, Inc., Catalog Number: AK103663, Purity > 95%). Methyl piperazine-2-carboxylate (IX) (ChemDiv, Inc., Catalog Number: FF20-0374, Purity > 90%). Ethyl 2-oxopiperidine-3-carboxylate (X) (Enamine, Ltd., Catalog Number Z397585734, Purity > 90%).

Cell lines.

ACH-2 and TZM-bl cell lines were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: ACH-257, 58 from Dr. Thomas Folks and TZM-bl59, 60, 61 , 62, 63 from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. ACH-2 cells were grown in Roswell Park Memorial Institute medium 1640 (RPMI 1640) supplemented with 25 mM HEPES, 0.3 g/L L-Glutamine, 10% heat-inactivated fetal bovine serum (FBS) and Pen/Strep. TZM-bl cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS and Pen/Strep. All of the cells were grown at 37 °C in the presence of 5% CO2.

Example 2. Methods

Here, 2* 10⁵ ACH-2 cells were seeded in 200 pL (1 microlitre = 10^-6 liters) on a 96-well plate, and HIV-1 provirus activation and infectious virus production was induced by the addition of 100 nM phorbol myristate acetate (PMA). The induced cells were incubated for 48 h with added compounds at given concentrations (0.5% DMSO was used as a vehicle control), and subsequently, the HIV-1 containing media was collected. The amount of HIV-1 p24 protein that was released into the media was measured using HIV1 p24 ELISA Kit (Abeam pic).

TZM-bl cells contain gene encoding for firefly luciferase (Luc) marker under control of HIV-1 LTR promoter. The expression of this marker is activated by the tat-protein produced by integrated HIV-1 provirus (Derdeyn, et al. 2000, Platt, et al. 1998, Kappes et al, 2004). In this assay, TZM-bl cells were seeded on a 96-well plate (2* 10⁵ cells per well). The next day, 50 pi (microlitre) of the supernatant of the incubation media containing virus treated with the compound at 10 mM concentration and polybrene (6pg/ml (6 microgram/millilitre) was added in the new plate with TZM-bl cells. The plate was incubated 2h at 37 °C and 150 mI DMEM medium was added to each well. The cells were thereafter incubated for 48 h at 37 °C. The supernatant was removed and the 50 mI of the lysis buffer was added to each well. The wells were then incubated 15 min and irradiated with UV light for 5 min at 254 nm wave length (to eliminate active virus). Then the Luc activity, which is in direct correlation with the amount of infectious virus used for infection of TZM-bl cells, was measured in cell lysates using Luciferase Assay System and Glomax 20/20 Luminometer (Promega) using 4 mI of lysate and 20 mI of the substrate (0.5% DMSO was used as a vehicle control).

Example 3. Effect of RNA methyltransferase complex METTL3/METTL14/WTAP activators on the HIV-1 provirus activation and virus replication.

The effect of the METTL3/METTL14/WTAP activators on the HIV-1 provirus activation and virus replication was measured using p24 ELISA assay. The increase in the virus production depends on the concentration of the activator compounds (VII), (IX) and (X) (Figures 1 (a), 1 (b) and 1 (c), respectively). For the three active compounds, a notable increase in the provirus activation and virus replication was detected at 10 nM concentration.

Example 4. Increase of the HIV-1 provirus activation and virus replication measured usinq virions oriqinatinq from cells pre-treated with the RNA methyltransferase

The aliquots of the supernatant from the cells treated with the compounds at 1 mM concentration were thereafter added to the plates with TZM-bl cells containing gene encoding for firefly luciferase marker under control of HIV-1 LTR promoter and virus growth medium (cf. Methods). After incubation for 48 h at 37 °C, the luciferase activity, which is in direct correlation with the amount of infectious virus used for infection of TZM-bl cells, was measured in cell lysates using the luciferase assay system. A substantial increase in the virus production was observed when the virus aliquots treated with previously observed active compounds (VII), (IX) and (X) were added to the growth medium (as compared to the control, no compound added).

References Cited

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Claims

1. A method of stimulating the HIV-1 provirus activation and subsequent replication by using a compound comprising agonists of RNA adenosine N-6 methylation.

2. The method of claim 1 , wherein the compounds comprising agonists of RNA adenosine N-6 methylation are activators of the RNA methyltransferase

M ETTL3/M ETTL 14/WTAP complex.

3. The method of claim 2, wherein the compound has a structure of Formula (I)

4. The method of claim 2, wherein the compound has a structure of Formula (II)

5. The method of claim 2, wherein the compound has a structure of Formula (II) wherein: R1 , R2, R3 and R4 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.

6. The method of claim 2, wherein the compound has a structure of Formula (IV)

7. The method of claim 2, wherein the compound has a structure of Formula (V)

wherein: R1 , R2, R3 and R4 are independently selected from the group consisting of FI, alkyl, aryl, aralkyl, acyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, alkylcarbamoyl, and dialkylcarbamoyl, aminoalkyl, aminoalaryl; or a pharmaceutically acceptable salt thereof.

8. The method of claim 2, wherein the compound has a structure of Formula (VI) wherein: R1 , R2, R3 and R4 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.

9. The method of claim 2, wherein the compound has a structure of Formula (VII)

10. The method of claim 2, wherein the compound has a structure of Formula (VIII)

11. The method of claim 2, wherein the compound has a structure of Formula (IX)

12. The method of claim 2, wherein the compound has a structure of Formula (X)

13. A pharmaceutical composition comprising the compound of any one of claims 2- 11 and a pharmaceutically acceptable excipient.

14. A vaccine composition comprising the compound of any one of claims 2-11 , a vaccine adjuvant, and an immunogenic agent.