Chemical Inhibitors Targeting the Oncogenic m⁶A Modifying Proteins

Abstract

Metrics & MoreArticle Recommendations CONSPECTUS: Epigenetics is brought to RNA, introducing a new dimension to gene expression regulation. Among numerous RNA modifications, N 6 -methyladenosine (m 6 A) is an abundant internal modification on eukaryote mRNA first identified in the 1970s. However, the significance of m 6 A modification in mRNA had been long neglected until the fat mass and obesity-associated (FTO) enzyme was identified as the first m 6 A demethylase almost 40 years later. The m 6 A modification influences nearly every step of RNA metabolism and thus broadly affects gene expression at multiple levels, playing a critical role in many biological processes, including cancer progression, metastasis, and immune evasion. The m 6 A level is dynamically regulated by RNA epigenetic machinery comprising methyltransferases such as methyltransferase-like protein 3 (METTL3), demethylases FTO and AlkB human homologue 5 (ALKBH5), and multiple reader proteins. The understanding of the biology of RNA epigenetics and its translational drug discovery is still in its infancy. It is essential to further develop chemical probes and lead compounds for an in-depth investigation into m 6 A biology and the translational discovery of anticancer drugs targeting m 6 A modifying oncogenic proteins.In this Account, we present our work on the development of chemical inhibitors to regulate m 6 A in mRNA by targeting the FTO demethylase, and the elucidation of their mode of action. We reported rhein to be the first substrate competitive FTO inhibitor. Due to rhein's poor selectivity, we identified meclofenamic acid (MA) that selectively inhibits FTO compared with ALKBH5. Based on the structural complex of MA bound with FTO, we designed MA analogs FB23-2 and Dac51, which exhibit significantly improved activities compared with MA. For example, FB23-2 is specific to FTO inhibition in vitro among over 400 other oncogenic proteins, including kinases, proteases, and DNA and histone epigenetic proteins. Mimicking FTO depletion, FB23-2 promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cells and inhibits the progression of primary cells in xenotransplanted mice. Dac51 treatment impairs the glycolytic activity of tumor cells and restores the function of CD8 + T cells, thereby inhibiting the growth of solid tumors in vivo. These FTO inhibitors were and will continue to be used as probes to promote biological studies of m 6 A modification and as lead compounds to target FTO in anticancer drug discovery.Toward the end, we also include a brief review of ALKBH5 demethylase inhibitors and METTL3 methyltransferase modulators.Collectively, these small-molecule modulators that selectively target RNA epigenetic proteins will promote in-depth studies on the regulation of gene expression and potentially accelerate anticancer target discovery.

Atomic Mutagenesis of N6-Methyladenosine Reveals Distinct Recognition Modes by Human m6A Reader and Eraser Proteins

“…The design of chemical inhibitors requires knowledge of the specific interactions between RNA and proteins. Such insights can be derived from crystal structures in combination with molecular dynamics simulations , and are supported by biochemical data obtained with mutated proteins, in which key functional amino acids in the active site are exchanged, e.g., by replacement with alanine. Introducing smaller changes by exchanging single functional groups via incorporation of artificial amino acids is more challenging (e.g., via stop-codon suppression).…”

“…Both enzymes belong to the family of nonheme iron­(II)- and α-ketoglutarate-dependent dioxygenases but remove the N 6 -methyl group via distinct reaction pathways: while demethylation by ALKBH5 directly yields adenosine accompanied by release of formaldehyde, , the oxidized intermediates N 6 -hydroxymethyladenosine (hm 6 A) and N 6 -formyladenosine (f 6 A) have been observed in the FTO-mediated pathway (Figure A). , While ALKBH5 has been observed to remove only internal m 6 A, preferably in DRACH motifs, FTO has also been shown to act on m 6 A m , m 1 A, m 3 U, and m 3 T. − The dysregulation of m 6 A and associated writer, reader, and eraser proteins has been connected to tumor development and disease. Therefore, m 6 A-modifying proteins have gained increasing attention as targets for drug development . The design of chemical inhibitors requires knowledge of the specific interactions between RNA and proteins.…”

A covalent compound selectively inhibits RNA demethylase ALKBH5 rather than FTO

“…Although 2OG analogs such as IOX3 14 and citrate 15 and other inhibitors such as MV1035, 16 ALK-04, 17 cmp-3, cmp-6, 18 20m, 19 and Ena15, 20 generally exhibit good inhibitory activity on ALKBH5 demethylation in vitro , there still lacks highly selective and potent ALKBH5 inhibitors in vivo . 21 Moreover, the inhibitory activities of these inhibitors on tumor cells are far from satisfactory. The tumor cells treated with ALKBH5 inhibitors did not exhibit the same phenotype as ALKBH5-knockdown cells.…”

Structure‐Activity Relationships of 2‐(Arylthio)benzoic Acid FTO Inhibitors

“…In this context, our long‐term aim is chemical intervention and target druggability validation of FTO [8] . In 2012, we reported rhein as the first FTO inhibitor [9] and developed a proof of concept to demonstrate that the level of m 6 A could be regulated by chemical small molecules, and researchers have since been interested in developing FTO inhibitors [10a,b] . The currently reported FTO inhibitors can be classified into four categories according to the mechanism of recognition: (A) substrate‐competitive inhibitors, such as rhein, [9] meclofenamic acid (MA), [11] N ‐CDPCB, [12] fluorescein, [13] CHTB, [14] FB23/FB23‐2, [7] Dac51, [15] Dac85, [16] ZLD115, [17] FTO‐4, [18] and TR‐FTO‐43N; [19] (B) 2‐OG‐competitive inhibitors, such as R ‐2HG; [20] (C) substrate/2‐OG dual competitive inhibitors, such as entacapone [21] and HZ‐MA hybrid; [22] and (D) inhibitors with unverified mechanisms, such as CS1/CS2, [23] saikosaponin D, [24] and rhein derivative (Figure S1) [25]…”