Fig. 1: Inhibitor-FAD adduct fragmentation proceeds through a Grob Mechanism.

a Chemical structures of LSD1 inhibitors GSK-LSD1 (1), T-448 (2), and TAK-418 (3). b Schematic illustrating the two activities disrupted by active-site covalent LSD1 inhibitors. Upon GSK-LSD1 (1) binding, LSD1 demethylase activity is disrupted (left) and the LSD1-GFI1B complex is dissociated (right). Cartoon adapted from Waterbury et al. ¹³. c Schematic of T-448 (2) LSD1-inhibition mechanism where the initial covalent inhibitor-FAD adduct (4) fragments to N-formyl-FAD (5). Cartoon adapted from Waterbury et al. ¹³. d Binding curves showing fluorescence polarization (y-axis) for increasing concentrations of LSD1-CoREST (x-axis) in the presence of a fluorescently labeled GFI1B(2-10) peptide after treatment with DMSO, GSK-LSD1 (1), or T-448 (2) (n = 2 biological replicates). e LC-MS spectra of FAD adducts after extracting recombinant LSD1 treated with d-T448 (6) for 4 h. f Schematic of proposed mechanism of covalent FAD-inhibitor adduct fragmentation to N-formyl-FAD (5) and T-448 styrene (10) via a Grob fragmentation. g Formation of T-448 styrene (10) detected by LC-MS after extracting recombinant LSD1 treated with T-448 at the indicated time points (n = 2 biological replicates). h Kinetics of styrene formation for LSD1 treated with T-448 (2) or p-Br-T448 (11) analog. Styrene formation was quantified relative to an internal standard and standard curves from chemically synthesized styrene standards (n = 3 biological replicates). Results in (d–h) are representative of two independent experiments. Data in (g) are provided in Supplementary Data 1. Source data are provided as a Source Data file for (d–h).