タンパク質メチル基転移酵素METTL13に対する選択的化学プローブの生物学的評価および分子動力学シミュレーションによる発見

METTL13, a dual-functional methyltransferase harboring protein N-terminal methyltransferase (NTMT) activity, has emerged as a pivotal regulator in carcinogenesis, tumor progression, and patient prognosis, thereby garnering substantial interest as a promising therapeutic target for cancers. Despite its clinical significance, no specific chemical probes or inhibitors have been reported to date, highlighting an urgent need for drug discovery efforts targeting this enzyme. Herein, we employed the bisubstrate inhibitor strategy to develop the chemical probe NT32 for METTL13, which showed selective against a panel of methyltransferases. Comprehensive investigations into the structural and functional impacts of NT32 on METTL13 were conducted via molecular docking, molecular dynamics (MD) simulations, and multi-dimensional analyses. Computational modeling revealed that NT32 could occupy both the SAM/SAH-binding pocket and the peptide substrate-binding site of METTL13 simultaneously, establishing a bivalent binding mode. Notably, MD simulations unveiled significant enhancements in both positive and negative residue correlations within the NT32-METTL13 complex compared to the wild-type enzyme. Specifically, interactions between NT32 and critical residues (Gly503, Asn614, Gly644, Glu645) strengthened the connectivity of Met692 with its surrounding residues. This structural reorganization was accompanied by augmented correlated motions between the Gly501-Val511 region (loop_501-504-α2_505-511) and the Leu613-Leu620 segment (β5_613-615-loop_616-618-α6_619-620), culminating in a more compact and stable protein conformation. Furthermore, NT32 specifically targeted and stabilized METTL13 in cellular thermal shift assay. These findings not only provide mechanistic insights into NT32-METTL13 interactions but also pave the way for discovery of METTL13 probes or inhibitors.