Ligand-Induced Opening of a Cryptic Pocket in METTL14

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the main human enzyme that deposits the most abundant internal mRNA modification, N⁶-methyladenosine (m⁶A). In the heterodimeric complex, METTL3 acts as a catalytic subunit while METTL14 is involved in mRNA binding and complex stabilization. Here, we present the discovery of small-molecule ligands that bind to a cryptic pocket in METTL14 by protein crystallography. A comparative analysis of crystal structures revealed that the METTL14 cryptic pocket is closed in the apo structure of METTL3-14, and in the structures of METTL3-14 in the complex with the cosubstrate S-adenosyl-methionine (SAM) and a large number of SAM-competitive inhibitors. We first discovered compounds 1 and 2 that bind to both the SAM pocket in METTL3 and the cryptic pocket in METTL14. With this structural information, we designed compound 3 that binds only to the METTL14 cryptic pocket. Compound 3 does not inhibit the catalytic activity of METTL3-14 but can be used as an anchor for heterobifunctional molecules. We propose a route for its further development into heterobifunctional ligands, e.g., proteolysis targeting chimeras (PROTACs).