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Modulating m6A Levels with Small-molecule Inhibitors - Why Target RNA Modifications?
July 25, 2024
Table of Contents:
Introduction: Why Target RNA Modifications?
The N6-methyladenosine (m6A) internal modification of eukaryotic mRNA significantly impacts RNA metabolism and gene expression and, as such, forms one layer of regulation impacting multiple normal and disease-associated biological processes. The complex interactions between RNA methylases (METTL3/METTL14 and WTAP), reader proteins (YTHDF, YTHDC, IGF2BP, and eIF3), and RNA demethylases (FTO and ALKBH5) (Shei, Wei, and He, Zaccara, Ries, and Jaffrey, and Yang et al.) remain incompletely explored, and, as such, a deeper understanding of RNA methylation and the potential development of small-molecule modulators of involved factors holds enormous potential in driving forward research and the development of anti-cancer therapies (Deng et al., 2013 and Deng et al., 2018).
Researchers guided by Cai-Guang Yang (University of Chinese Academy of Sciences) recently reported on their ongoing structure-based approach to developing small-molecule inhibitors that regulate RNA methylation levels by targeting the m6A demethylase FTO (Fat mass and obesity-associated protein) (Jia et al.). Previous research revealed that the pharmacological inhibition of FTO suppressed tumor progression and improved outcomes in a mouse model (Cui et al.); therefore, they hoped that targeting m6A via ever-improving small-molecule FTO inhibitors could support basic epigenetic research and drive the development of advanced anti-cancer therapies at the same time.
The First Substrate-Competitive Small-molecule Inhibitor of FTO and Beyond
The Yang lab had previously reported rhein as the first reported substrate-competitive FTO inhibitor in 2012 (Chen et al.). While this major bioactive component of rhubarb displayed low cytotoxicity and induced a significant increase in m6A levels, the poor selectivity of rhein prompted side effects that interfered with its potential application in anti-cancer therapies (Li et al.); therefore, Huang et al. screened for compounds that competed with methylated nucleic acids to specifically bind FTO but not the ALKBH5 RNA demethylase (Huang et al., 2015). Additional validation following the identification of candidates identified meclofenamic acid (an anthranilic acid-class non-steroidal anti-inflammatory) as a specific substrate competitive inhibitor of FTO.
Based on a structural complex of meclofenamic acid bound to FTO, the authors subsequently designed FB23-2 (Huang et al., 2019) and Dac51 (Liu et al.) as meclofenamic acid analogs with significantly improved characteristics. FB23-2 specifically inhibited FTO (and not over 400 other oncogenic proteins, such as kinases, proteases, and epigenetic modification-associated proteins) and significantly induced m6A levels (Huang et al., 2019). When evaluated for its potential therapeutic use, FB23-2 promoted the differentiation/apoptosis of human cancer cells and inhibited the progression of primary tumor cells in Xeno transplanted mice and extended their lifespans but displayed minimal anti-proliferative effects on human normal cells. Similar studies with Dac51 revealed that treatment with this advanced small-molecule FTO inhibitor also induced an increase in m6A levels; however, Dac51 also impaired the glycolytic capacity of tumor cells and restored the function of CD8+ T lymphocytes to inhibit the growth of solid tumors in vivo but exhibited minimal toxicity on normal cells. Further development led to the design of 3a/Dac85, which induced increased m6A levels and displayed similar anti-cancer activities, and 44/ZLD115, which incorporated flexible alkaline side chains at FB23’s solvent-accessible positions (Xiao et al.) to create an inhibitor with more balanced physicochemical properties that increased m6A levels and displayed yet greater anti-cancer potential.
The Ongoing Application of Small-molecule Inhibitors of FTO
Finally, the authors reported on the application of these small-molecule FTO inhibitors in RNA-based epigenetics research, which helped to highlight the feasibility of suppressing tumor growth by inhibiting FTO. Briefly, rhein administration revealed how m6A modulated nervous system development (Yu et al.), treatment with an ethyl ester form of meclofenamic acid inhibited cancer stem cell growth (Cui et al.), while Dac51 exposure significantly impaired uterine leiomyosarcoma proliferation (Yang et al.). FB23-2 administration helped to identify LINE1 RNA as a primary substrate of FTO in mouse embryonic stem cells (Wei et al.), revealed how FTO inhibition blocked adipogenesis (Wang et al.), suppressed pancreatic cancer growth (Dong et al.), and altered tumor responsiveness to radiotherapy in combination with a ferroptosis activator in a nasopharyngeal carcinoma cell model (Huang et al.).
The Next Big Steps for Small-molecule Inhibitors
The authors hope that their body of work identifying highly selective and potent small-molecule inhibitors of FTO through a structure-based rational design approach will lead to additional findings regarding the epigenetic regulation of gene expression through RNA methylation and propel the development of anti-cancer therapies and synergistic approaches that mitigate drug resistance and enhance therapeutic efficacy.
For more on how targeting m6A levels via small-molecule inhibitors can support basic epigenetics research and drive anti-cancer therapy development, see Accounts of Chemical Research, October 2023.
About the author
Stuart P. Atkinson, Ph.D.
Stuart was born and grew up in the idyllic town of Lanark (Scotland). He later studied biochemistry at the University of Strathclyde in Glasgow (Scotland) before gaining his Ph.D. in medical oncology; his thesis described the epigenetic regulation of the telomerase gene promoters in cancer cells. Following Post-doctoral stays in Newcastle (England) and Valencia (Spain) where his varied research aims included the exploration of epigenetics in embryonic and induced pluripotent stem cells, Stuart moved into project management and scientific writing/editing where his current interests include polymer chemistry, cancer research, regenerative medicine, and epigenetics. While not glued to his laptop, Stuart enjoys exploring the Spanish mountains and coastlines (and everywhere in between) and the food and drink that it provides!
Contact Stuart on Twitter with any questions
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