Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5

Huang, Yue; Yan, Jingli; Li, Qi; Li, Jiafei; Gong, Shouzhe; Zhou, Hu; Gan, Jianhua; Jiang, Hualiang; Jia, Guifang; Luo, Cheng; Yang, Cai‐Guang

doi:10.1093/nar/gku1276

Cited by 468 publications

(483 citation statements)

References 65 publications

Supporting

Mentioning

470

Contrasting

Order By: Relevance

“…The IC 50 was quantitatively determined to be 12.7 M in the HPLC-based detection when assayed at 50 M 2OG (Fig. 1D) (48). BA is a moderate inhibitor of FTO (Fig.…”

Section: Resultsmentioning

confidence: 99%

Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage

Huang

Liu

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The AlkB repair enzymes, including Escherichia coli AlkB and two human homologues, ALKBH2 and ALKBH3, are iron(II)-and 2-oxoglutarate-dependent dioxygenases that efficiently repair N 1 -methyladenine and N 3 -methylcytosine methylated DNA damages. The development of small molecule inhibitors of these enzymes has seen less success. Here we have characterized a previously discovered natural product rhein and tested its ability to inhibit AlkB repair enzymes in vitro and to sensitize cells to methyl methane sulfonate that mainly produces N 1 -methyladenine and N 3 -methylcytosine lesions. Our investigation of the mechanism of rhein inhibition reveals that rhein binds to AlkB repair enzymes in vitro and promotes thermal stability in vivo. In addition, we have determined a new structural complex of rhein bound to AlkB, which shows that rhein binds to a different part of the active site in AlkB than it binds to in fat mass and obesity-associated protein (FTO). With the support of these observations, we put forth the hypothesis that AlkB repair enzymes would be effective pharmacological targets for cancer treatment.The nucleic acids in living cells are subject to modification by both endogenous and environmental agents (1). Direct-acting chemicals constantly damage nucleic acids and generate various methyl lesions with mutagenic and/or cytotoxic consequences (2, 3). O 6 -Methylguanine (O 6 mG) 2 and N 3 -methyladenine lesions have the highest potential for methylating damage by an SN1 agent such as N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG), which block replication and are thought to be toxic (4, 5). For the most part, the SN2 agent such as methyl methane sulfonate (MMS) produces N 1 -methyladenine (m 1 A) and N 3 -methylcytosine (m 3 C) lesions in single-stranded DNA (ssDNA). Accumulation of these adducts can lead to cell death (6, 7). Organisms have evolved several mechanisms to efficiently remove various methyl lesions, including suicidal methyltransferases, DNA glycosylases, and the AlkB family dioxygenases (see Fig. 1A) (8, 9). To date, AlkB repair appears to be the major natural defense mechanism with the power to restore the canonical base structure in vivo. Escherichia coli AlkB and its human homologues, ALKBH2 and ALKBH3, utilize iron(II) and 2-oxoglutarate (2OG) to achieve oxidative demethylation of m 1 A and m 3 C (see Fig. 1B) (10 -12). The lack of AlkB repair results in increased sensitivity to MMS, elevated level of mutations, and reduced cell proliferation (13-16). Furthermore, the accumulation of m 1 A and m 3 C lesions could also occur on RNA. Research suggests that the oxidative demethylation in mRNA and tRNA acts as a part of AlkB or ALKBH3 repair to protect cells against MMS (17, 18). The scope of substrates for AlkB repair has been largely extended to all simple N-alkyl lesions at the WatsonCrick base-pairing interface on the four bases (19), thus indicating the importance of oxidative demethylation for cell survival. In addition, human enzymes have been broadly linked to cancer. The housekeeping ...

show abstract

“…The IC 50 was quantitatively determined to be 12.7 M in the HPLC-based detection when assayed at 50 M 2OG (Fig. 1D) (48). BA is a moderate inhibitor of FTO (Fig.…”

Section: Resultsmentioning

confidence: 99%

Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage

Huang

Liu

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The natural product rhein was reported as an inhibitor of FTO with an IC 50 value of 30 µM using m 6 A-containing 15-mer ss-RNA as substrate and a high-performance liquid chromatography (HPLC)-based assay. 31 Huang et al 32 identified meclofenamic acid (MA) as a highly selective inhibitor of FTO (IC 50 : 8 µM) over ALKBH5 (no inhibition) using HPLC-based assays. 32 They also used a fluorescence polarization-based assay to test the displacement of ssDNA by MA.…”

Section: Discussionmentioning

confidence: 99%

A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5

Kennedy

Hajian

et al. 2016

SLAS Discovery

View full text Add to dashboard Cite

N6 -methyladenosine (m 6 A) is the most common reversible internal modification in mammalian RNA. Changes in m 6 A levels have been implicated in a variety of cellular processes, including nuclear RNA export, control of protein translation, and protein splicing, and they have been linked to obesity, cancer, and other human diseases. METTL3 and METTL14 are N 6 -adenosine methyltransferases that work more efficiently in a stable METTL3-METTL14 heterodimer complex (METTL3-14). ALKBH5 is an m 6 A-RNA demethylase that belongs to the AlkB family of dioxygenases. We report the development of radioactivity-based assays for kinetic characterization of m 6 A-RNA modifications by METTL3-14 complex and ALKBH5 and provide optimal assay conditions suitable for screening for ligands in a 384-well format with Z′ factors of 0.78 and 0.77, respectively.

show abstract

“…As alternative approaches to further investigate the effects of m 6 A on KSHV lytic gene expression, we next attempted to increase m 6 A by treating BCBL1 cells with meclofenamic acid (MA), a selective inhibitor of FTO (70), or block m 6 A with DAA. We first determined the cytotoxicity and optimal concentrations of MA and DAA by treating BCBL1 cells with various concentrations of these reagents for 24 h, followed by propidium iodide (PI) staining of the cells and flow cytometry analysis of cell viability.…”

mentioning

confidence: 99%

Kaposi's Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N ⁶ -Adenosine Methylation To Promote Lytic Replication

Chen

Nilsen

2017

J Virol

119

133

View full text Add to dashboard Cite

N 6 -adenosine methylation (m 6 A) is the most common posttranscriptional RNA modification in mammalian cells. We found that most transcripts encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) genome undergo m 6 A modification. The levels of m 6 A-modified mRNAs increased substantially upon stimulation for lytic replication. The blockage of m 6 A inhibited splicing of the pre-mRNA encoding the replication transcription activator (RTA), a key KSHV lytic switch protein, and halted viral lytic replication. We identified several m 6 A sites in RTA pre-mRNA crucial for splicing through interactions with YTH domain containing 1 (YTHDC1), an m 6 A nuclear reader protein, in conjunction with serine/arginine-rich splicing factor 3 (SRSF3) and SRSF10. Interestingly, RTA induced m 6 A and enhanced its own premRNA splicing. Our results not only demonstrate an essential role of m 6 A in regulating RTA pre-mRNA splicing but also suggest that KSHV has evolved a mechanism to manipulate the host m 6 A machinery to its advantage in promoting lytic replication.IMPORTANCE KSHV productive lytic replication plays a pivotal role in the initiation and progression of Kaposi's sarcoma tumors. Previous studies suggested that the KSHV switch from latency to lytic replication is primarily controlled at the chromatin level through histone and DNA modifications. The present work reports for the first time that KSHV genome-encoded mRNAs undergo m 6 A modification, which represents a new mechanism at the posttranscriptional level in the control of viral replication.KEYWORDS KSHV, N 6 -adenosine methylation, RNA splicing, lytic replication G ene expression is controlled not only at the chromatin level through histone and DNA modifications but also at the posttranscriptional level through RNA modifications. N 6 -adenosine methylation (m 6 A) is the most abundant RNA modification found in ϳ25% of RNA species in mammalian cells (1, 2). Despite its discovery decades ago (3-7), the biochemical pathways responsible for m 6 A and the biological functions of this process were not fully defined until very recently (8-10). Three methyltransferases, including methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms' tumor 1-associated protein (WTAP), act as m 6 A writers and catalyze RNA m 6 A at specific sites with the consensus sequence [(G/A)GAC, where the underlined adenosine is the methylation site] (11, 12). Two demethylases, fat mass-and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5), both of which act as m 6 A erasers, reverse this process (13-17). Most m 6 A sites are located near the transcription start sites, exonic regions flanking splicing sites, stop codons, and the 3= untranslated region (3= UTR) (1,

show abstract

Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5

Cited by 468 publications

References 65 publications

Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage

Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage

A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5

Kaposi's Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N ⁶ -Adenosine Methylation To Promote Lytic Replication

Contact Info

Product

Resources

About

Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5

Cited by 468 publications

References 65 publications

Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage

Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage

A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5

Kaposi's Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N 6 -Adenosine Methylation To Promote Lytic Replication

Contact Info

Product

Resources

About

Kaposi's Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N ⁶ -Adenosine Methylation To Promote Lytic Replication