Nucleic Acids Res. 2015 Jan; 43(1):373-84. doi: 10.1093/nar/gku1276.

Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5.

Yue Huang,Jingli Yan, Qi Li, Jiafei Li, Shouzhe Gong, Hu Zhou, Jianhua Gan, Hualiang Jiang, Gui-Fang Jia, * Cheng Luo,* and Cai-Guang Yang*

 

*Correspondence:

Cai-Guang Yang, PhD

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

yangcg@simm.ac.cn

 

Abstract

Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and ALKBH5, oxidatively demethylate abundant N(6)-methyladenosine (m6A) residues in mRNA. Achieving a method for selective inhibition of FTO over ALKBH5 remains a challenge, however. Here, we have identified meclofenamic acid (MA) as a highly selective inhibitor of FTO. MA is a non-steroidal, anti-inflammatory drug that mechanistic studies indicate competes with FTO binding for the m6A-containing nucleic acid. The structure of FTO/MA has revealed much about the inhibitory function of FTO. Our newfound understanding, revealed herein, of the part of the nucleotide recognition lid (NRL) in FTO, for example, has helped elucidate the principles behind the selectivity of FTO over ALKBH5. Treatment of HeLa cells with the ethyl ester form of MA (MA2) has led to elevated levels of m6A modification in mRNA. Our collective results highlight the development of functional probes of the FTO enzyme that will (i) enable future biological studies and (ii) pave the way for the rational design of potent and specific inhibitors of FTO for use in medicine.

PMID: 25452335

 

 

Supplementary

As a severe metabolic syndrome affecting over 500 million people worldwide, obesity is brings with it an increased risk to several co-morbidities, including type 2 diabetes, cardiovascular disorders, and cancer. Genome-wide association studies have revealed associations between the fat mass and obesity-associated transcript1. Animal model showed that the inactivation of FTO protected from obesity2, which indicates that FTO might be also involved in human energy homeostasis in a significant way. Indeed, mutation of FTO impacts about 1 billion members of the human population. Very recently, FTO was shown to functionally relate to adipogenesis in mouse 3T3-L1 cell lines3. Therefore, small-molecule inhibitors of FTO should be developed with the aim of eventually producing threapies that target FTO for obesity and diabetes.

 

The FTO enzyme has been identified as the first m6A demethylase of mRNA, which is involved in the dynamic regulation of the cellular levels of endogenous m6A modification on mRNA4. Besides the catalytic demethylation of m6A in vivo, FTO is involved in a number of protein-protein interactions5. The genetic knockdown of FTO would disturb not only m6A demethylation but also these important PPIs. Thus, while the genetic methods are powerful, they are ultimately complementary to pharmacological methods for understanding protein function and therapeutic utility. Therefore, specific inhibitor of FTO demethylation would be extremely useful for biological studies of RNA epigenetics. Such a small-molecule inhibitor of FTO demethylation would enable temporal regulation of the specific m6A residues and also facilitate studies of FTO target specificity and function. To this end, several small-molecule inhibitors targeting FTO demethylation have been developed. The first cell-active inhibitor of FTO is the natural compound rhein, which was identified by the structure-based virtual screening6. The enzyme kinetics and structural analyses have identified rhein as a competitive inhibitor of nucleic acid substrates. Recently, a strategy of 2OG-tethering, which simultaneously occupies both 2OG co-factor and the substrate binding site, has been successfully utilized for the development of selective inhibitor of FTO demethylation7; however, the cellular 2OG might poison the potency of these 2OG derivatives. In this highlight paper, we have demonstrated that an old drug, meclofenamic acid, can selectively inhibit FTO demethylaiton over ALKBH5, which is the second m6A demethylase. Identified by a high-throughput fluorescence polarization assay, MA is outstanding from the screening of a library of old drugs. The inhibitor mode of action study has identified a unique binding site for the specific inhibition of FTO demethylation over other dioxygenases. Now, our work focuses on the optimization of the MA compound to block the off-target effects and to improve cellular potency and eventually develop specific functional probes that target FTO for biological and therapeutic purposes.

 

Ideological changes in our understanding of obesity are characterized by a state of chronic long-term inflammation. Obese individuals have higher levels of inflammatory factors produced by adipocytes, including TNF-α,interleukin-1 or interleukin-6. Of note, MA is a non-steroidal anti-inflammatory drug, primarily known for its inhibition of cyclooxygenases and lipoxygenases to disrupt the synthesis of prostaglandins. MA is of great interest because the recently identified FTO inhibitors are also derived from dual cyclooxygenase/lipoxygenase inhibitors8. Any possible connections between anti-inflammatory and selective inhibition of FTO demethylation remains to be explored. Such studies might even establish relevance between the regulation of m6A modification and the gene expression relative to obesity or diabetes. In summary, these studies arise alongside increasing interest in the exploration of genetic epidemiology and the complex biology of FTO as well as the development of therapies for obesity and diabetes by targeting FTO demethylation.

 

Importance of the study

Our work sheds light on the development of new chemical probes for studying the roles of human m6A demethylases in RNA epigenetic processes and presents possibilities for providing therapeutic leads for drug discovery in the field of obesity and diabetes.

 

References

1              Frayling, T. M. et al. A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science 316, 889-894, doi:10.1126/science.1141634 (2007).

2              Fischer, J. et al. Inactivation of the Fto gene protects from obesity. Nature 458, 894-898, doi:10.1038/nature07848 (2009).

3              Zhao, X. et al. FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis. Cell Res 24, 1403-1419, doi:10.1038/cr.2014.151 (2014).

4              Jia, G. et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol 7, 885-887, doi:10.1038/nchembio.687 (2011).

5              Gulati, P. et al. Role for the obesity-related FTO gene in the cellular sensing of amino acids. Proceedings of the National Academy of Sciences of the United States of America 110, 2557-2562, doi:10.1073/pnas.1222796110 (2013).

6              Chen, B. et al. Development of cell-active N6-methyladenosine RNA demethylase FTO inhibitor. J Am Chem Soc 134, 17963-17971, doi:10.1021/ja3064149 (2012).

7              Toh, J. D. W. et al. A strategy based on nucleotide specificity leads to a subfamily-selective and cell-active inhibitor of N6-methyladenosine demethylase FTO. Chemical Science 6, 112-122, doi:10.1039/C4SC02554G (2015).

8              Zheng, G. et al. Synthesis of a FTO inhibitor with anticonvulsant activity. ACS Chem Neurosci 5, 658-665, doi:10.1021/cn500042t (2014).

 

Acknowledgements:

This study was supported by National Natural Science Foundation of China [91313303, 21372237, 21372022, and 21210003], National Basic Research Program [2015CB910603], Hi-Tech Research and Development Program of China [2012AA020302].

 

 

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