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.

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Human ABCB6 is an ATP-binding cassette transporter that regulates heme biosynthesis by translocating various porphyrins from the cytoplasm into the mitochondria. Here we report the cryo-electron microscopy (cryo-EM) structures of human ABCB6 with its substrates, coproporphyrin III (CPIII) and hemin, at 3.5 and 3.7 Å resolution, respectively. Metalfree porphyrin CPIII binds to ABCB6 within the central cavity, where its propionic acids form hydrogen bonds with the highly conserved Y550. The resulting structure has an overall fold similar to the inward-facing apo structure, but the two nucleotide-binding domains (NBDs) are slightly closer to each other. In contrast, when ABCB6 binds a metal-centered porphyrin hemin in complex with two glutathione molecules (1 hemin: 2 glutathione), the two NBDs end up much closer together, aligning them to bind and hydrolyze ATP more efficiently. In our structures, a glycine-rich and highly flexible "bulge" loop on TM helix 7 undergoes significant conformational changes associated with substrate binding. Our findings suggest that ABCB6 utilizes at least two distinct mechanisms to fine-tune substrate specificity and transport efficiency.