Tideglusib as a First-in-Class Chemical Probe for NatA: Identification, Mechanism, and Cellular Validation of an...

N-terminal acetyltransferase A (NatA) catalyzes 40-57% of eukaryotic Nα-acetylation, a co-translational modification critical for protein stability, localization, and function, yet no validated small-molecule probe of NatA has been reported. Here, we describe the discovery and characterization of tideglusib (YD3139), a clinical-stage GSK-3β inhibitor, as the first small-molecule probe for NatA through a unprecedented catalysis-dependent mechanism. This dual-event mechanism was established through intact protein MS, rapid dilution assays, and substrate-independent IC50 profiling. Tideglusib binds allosterically to Saccharomyces cerevisiae NatA (yNatA), whereupon the CoA released during catalysis chemically modifies tideglusib to form a reversible covalent adduct, a new paradigm for acetyltransferase inhibition. Tideglusib was identified by screening of an in-house library (∼700 compounds) and confirmed by structural optimization of the thiadiazolidinedione scaffold, yielding IC50 values of 0.68-1.8 μM against yeast and human NatA with >20-fold selectivity over other NAT family members. Quantitative proteomics confirmed on-target suppression of NatA-mediated Nα-acetylation in yeast. These findings establish tideglusib as a mechanistic chemical probe for NatA, reveal NatA as a previously unrecognized molecular target for a promiscuous clinical-stage investigational compound, and introduce a conceptual framework for exploiting catalysis-dependent adduct formation as a new strategy for acetyltransferase inhibitor design.