Eltrombopag as an Allosteric Inhibitor of the METTL3-14 Complex Affecting the m6A Methylation of RNA in Acute Myeloid Leukemia Cells

Abstract

N6A-methyladenosine (m6A) post-transcriptional modification, the most abundant internal RNA modification, is catalyzed by the METTL3-14 methyltransferase complex. Recently, attention has been drawn to the METTL3-14 complex regarding its significant roles in the pathogenesis of acute myeloid leukemia (AML), attracting the potential of novel therapeutic targets for the disease. Herein, we report the identification and characterization of eltrombopag as a selective allosteric inhibitor of the METTL3-14 complex. Eltrombopag exhibited selective inhibitory activity in the most active catalytic form of the METTL3-14 complex by direct binding, and the mechanism of inhibition was confirmed as a noncompetitive inhibition by interacting at a putative allosteric binding site in METTL3, which was predicted by cavity search and molecular docking studies. At a cellular level, eltrombopag displayed anti-proliferative effects in the relevant AML cell line, MOLM-13, in correlation with a reduction in m6A levels. Molecular mechanism studies of eltrombopag using m6A-seq analysis provided further evidence of its cellular function by determining the hypomethylation of leukemogenic genes in eltrombopag-treated MOLM-13 cells and the overlapping of the pattern with those of METTL3-knockdown MOLM-13 cells. In conclusion, eltrombopag was first disclosed as a functional METTL3-14 allosteric inhibitor in AML cells, which could be utilized for the further development of novel anti-AML therapy.

Methyladenosine Modification in RNAs: From Regulatory Roles to Therapeutic Implications in Cancer

“…More recently, Yankova et al, have identified a selective METTL3 catalytic inhibitor, STM2457, which could suppress the growth of AML [163]. Lee et al, reported that eltrombopag selectively inhibits the catalytic form of the METTL3-14 complex that can decrease the m 6 A modification on RNA in AML cells [164]. Meanwhile, two FTO inhibitors (FB23 and FB23-2) have been designed by Huang et al, exhibiting a selective inhibitory effect on the proliferation of AML [165].…”

METTL3 from Target Validation to the First Small-Molecule Inhibitors: A Medicinal Chemistry Journey

“…Moreover, 11a forms extensive van der Waals interactions with aromatic residues and hydrophobic amino acids such as Val452, Val485, and Val487. 145 The importance of the carboxylic acid for the METTL3−11a interaction was confirmed by the massive drop in inhibitory potency caused by the removal or esterification of the carboxylic acid (compound 11b or 11c, respectively). Similarly, the removal of the phenolic hydroxyl (11d) group caused a fourfold decrease in potency (Figure 7B).…”

“…According to docking studies, 11a binds to an allosteric binding site distinct from the SAM pocket, with the carboxylic acid moiety forming hydrogen bonds with the backbone amides of Asp499 and Cys550, while the phenol forms a hydrogen bond with the carboxylate group of Asp453 and the hydrazine moiety forms a hydrogen bond with the carboxamide group of Gln496. Moreover, 11a forms extensive van der Waals interactions with aromatic residues and hydrophobic amino acids such as Val452, Val485, and Val487 . The importance of the carboxylic acid for the METTL3– 11a interaction was confirmed by the massive drop in inhibitory potency caused by the removal or esterification of the carboxylic acid (compound 11b or 11c , respectively).…”

“…The importance of the carboxylic acid for the METTL3– 11a interaction was confirmed by the massive drop in inhibitory potency caused by the removal or esterification of the carboxylic acid (compound 11b or 11c , respectively). Similarly, the removal of the phenolic hydroxyl ( 11d ) group caused a fourfold decrease in potency (Figure B) . Compound 11a was then tested in a cellular context, where it inhibited the AML MOLM-13 cell line growth (GI 50 = 8.28 μM), and dose-dependently reduced the m 6 A levels after 24 h of treatment.…”

“…Similarly, the removal of the phenolic hydroxyl ( 11d ) group caused a fourfold decrease in potency ( Figure 7 B). 145 Compound 11a was then tested in a cellular context, where it inhibited the AML MOLM-13 cell line growth (GI 50 = 8.28 μM), and dose-dependently reduced the m 6 A levels after 24 h of treatment. Moreover, 11a displayed synergistic antiproliferative activity when tested in MOLM-13 cells in combination with the AML approved drug venetoclax, known BCL-2 inhibitor, while weak or no synergy was observed in combination with other AML drugs such as gliterinib, cytarabine, and sorafenib.…”