Covalent adduct Grob fragmentation underlies LSD1 demethylase-specific inhibitor mechanism of action and resistance

Amanda L. Waterbury, Jonatan Caroli, Olivia Zhang, Paloma R. Tuttle, Chao Liu, Jiaming Li, Ji Sung Park, Samuel M. Hoenig, Marco Barone, Airi Furui, Andrea Mattevi & Brian B. Liau

Nat Commun 16, 3156 (2025). 

https://doi.org/10.1038/s41467-025-57477-3

Chromatin modifiers often work in concert with transcription factors (TFs) and other complex members, where they can serve both enzymatic and scaffolding functions. Due to this, active site inhibitors targeting chromatin modifiers may perturb both enzymatic and nonenzymatic functions. For instance, the antiproliferative effects of active-site inhibitors targeting lysine-specific histone demethylase 1A (LSD1) are driven by disruption of a protein-protein interaction with growth factor independence 1B (GFI1B) rather than inhibition of demethylase activity. Recently, next-generation precision LSD1 covalent inhibitors have been developed, which selectively block LSD1 enzyme activity by forming a compact N-formyl flavin adenine dinucleotide (FAD) adduct that spares the GFI1B interaction. However, the mechanism accounting for N-formyl-FAD formation remains unclear. Here we clarify the mechanism of these demethylase-specific inhibitors of LSD1, demonstrating that the covalent inhibitor-FAD adduct undergoes a Grob fragmentation. Using inhibitor analogs and structural biology, we identify structure-activity relationships that promote this transformation. Furthermore, we unveil an unusual drug resistance mechanism whereby distal active-site mutations can promote inhibitor-adduct Grob fragmentation even for previous generation compounds. Our study uncovers the unique Grob fragmentation underlying the mechanism of action of precision LSD1 enzyme inhibitors, offering insight into their reactivity with broader implications for drug resistance.

Size-Dependent Target Engagement of Covalent Probes

László Petri, Ronen Gabizon, György G. Ferenczy, Nikolett Péczka, Attila Egyed, Péter Ábrányi-Balogh, Tamás Takács, and György M. Keserű

Journal of Medicinal Chemistry 2025 68 (6), 6616-6632

DOI: 10.1021/acs.jmedchem.5c00017

Labeling proteins with covalent ligands is finding increasing use in proteomics applications, including identifying nucleophilic residues amenable for labeling and in the development of targeted covalent inhibitors (TCIs). Labeling efficiency is measured by the covalent occupancy of the target or by biochemical activity. Here, we investigate how these observed quantities relate to the intrinsic parameters of complex formation, namely, noncovalent affinity and covalent reactivity, and to experimental conditions, including incubation time and ligand concentration. It is shown that target engagement is beneficially driven by noncovalent recognition for lead-like compounds, which are appropriate starting points for targeted covalent inhibitors owing to their easily detectable occupancy and fixed binding mode, facilitating optimization. In contrast, labeling by fragment-sized compounds is inevitably reactivity-driven as their small size limits noncovalent affinity. They are well-suited for exploring ligandable nucleophilic residues, while small fragments are less appropriate starting points for TCI development.

O-Cyanobenzaldehydes Irreversibly Modify Both Buried and Exposed Lysine Residues in Live Cells

Huan Ling, Lin Li, Liping Duan, Weixue Huang, Jiangnan Zheng, Shijie Zhang, Xinling Li, Xiaorong Qiu, Yang Zhou, Nan Ma, Xiaomei Ren, Jinwei Zhang, Zhen Wang, Yujun Zhao, Ruijun Tian, Zhi-Min Zhang, and Ke Ding

Journal of the American Chemical Society 2025

DOI: 10.1021/jacs.4c18006

Lysine residue represents an attractive site for covalent drug development due to its high abundance (5.6%) and critical functions. However, very few lysines have been characterized to be accessible to covalent ligands and perturb the protein functions, owing to their protonation state and adjacent steric hindrance. Herein, we report a new lysine bioconjugation chemistry, O-cyanobenzaldehyde (CNBA), that enables selective modification of the lysine ε-amine to form iso-indolinones under physiological conditions. Activity-based proteome profiling enabled the mapping of 3451 lysine residues and 85 endogenous kinases in live cells, highlighting its potential for modifying hyper-reactive lysines within the proteome or buried catalytic lysines within the kinome. Further protein crystallography and mass spectrometry confirmed that K271_ABL1 and K162_AURKA are covalently targetable sites in kinases. Leveraging a structure-based drug design, we incorporated CNBA into the core structure of Nutlin-3 to irreversibly inhibit the MDM2-p53 interaction by targeting an exposed lysine K94 on the surface of murine double minute 2. Importantly, we have demonstrated the potential application of CNBA as a lysine-recognized bioconjugation agent for developing new antibody-drug conjugates. The results collectively validate CNBA as a new selective and efficient modifying agent with broad applications for both buried and exposed lysine residues in live cells.