A Tandem Bioorthogonal Retro-Cope and Cope Elimination for the Activation of Covalent Inhibitors with an Acrylamide or Vinylsulfonamide Warhead in Live Cells

Yan Huang, Miao Liu, Dongguang Fan, Fan Xu, Fushuang Xiang, Qingqiang Min, and Xingyue Ji

Journal of the American Chemical Society 2026

DOI: 10.1021/jacs.6c00226

Precisely controlling the activation of covalent inhibitors through the caging and decaging of their reactive warheads is pivotal, yet this strategy is rarely pursued due to its formidable technical challenges. In this contribution, we report a novel tandem bioorthogonal retro-Cope and Cope elimination designed for efficient and selective activation of the covalent inhibitors bearing an acrylamide or vinylsulfonamide warhead in live cells. Notably, this strategy can be simultaneously tailored to coactivate both the covalent inhibitor and a fluorescent reporter, enabling real-time monitoring of prodrug activation. We successfully demonstrate the proof of concept through on-demand activation of two distinct EGFR covalent inhibitors and a BRD4-targeting molecular glue in live cells. This approach allows precise control over antiproliferative activity or induced protein degradation exclusively upon triggering via the tandem bioorthogonal reaction. We anticipate that this methodology will open new avenues for the selective delivery and controlled activation of covalent inhibitors, with broad potential in chemical biology and targeted therapy.

Discovery of a Potent, Selective, and In Vivo Efficacious Covalent Inhibitor for Lysine Methyltransferase SETD8

He Chen, Rudra Prasad Dutta, Zhizhong Li, Yue Zhong, Anqi Ma, Kwang-Su Park, Jithesh Kottur, Alison Park, Nicolas Babault, Ke Wang, Dandan Wang, Yan Xiong, H. Ümit Kaniskan, Minkui Luo, Samir Parekh, and Jian Jin

Journal of Medicinal Chemistry 2026

DOI: 10.1021/acs.jmedchem.5c02958

Dysregulated signaling of SET domain-containing protein 8 (SETD8) has been implicated in tumorigenesis, yet most SETD8 inhibitors exhibited limited cellular efficacy. Herein, we developed a potent and selective SETD8 covalent inhibitor, MS2928 (3), featuring a propiolamide covalent warhead. Compound 3 potently and selectively inhibited SETD8 methyltransferase activity. The covalent inhibition mechanism of 3 was confirmed by mass spectrometry and X-ray crystallography. Moreover, 3 significantly reduced the histone H4 lysine 20 monomethylation (H4K20me1) levels in cells and robustly inhibited the proliferation of SETD8-overexpressing multiple myeloma (MM) cell lines with no significant antiproliferative effect on SETD8-low expressing MM cells and normal cells. Importantly, 3 effectively inhibited tumor growth in vivo in two xenograft mouse models of SETD8-overexpressing MM cell lines. Collectively, our results establish 3 as a valuable chemical tool for exploring the biological functions of SETD8 and pave the way for further development of novel epigenetic therapies for MM.

Exploiting human fucosyltransferase 8 allostery with a covalent inhibitor for core fucosylation suppression

Jiheng Jiang, Dongyang He, Mengyu Ke, Jinhua Qin, Guang Yang, Biao Yu, Jing Wang & Pengfei Fang 

Nat Commun (2026). 

https://doi.org/10.1038/s41467-026-68971-7

Core fucosylation, catalyzed by fucosyltransferase 8 (FUT8), plays critical roles in cancer progression, immune evasion, and drug resistance, making it a compelling therapeutic target. However, development of selective FUT8 inhibitors has been hindered by shared substrate specificity of fucosyltransferases. Here, we report the discovery of a previously unrecognized allosteric site on FUT8 and the development of a low-toxicity covalent inhibitor, CAIF (stearic acid-N-hydroxysuccinimide ester-dimethylimidazolium bromide), through structure-based drug design. High-throughput screening and crystallographic studies reveal that small molecules such as NH125 bind to a channel-like allosteric pocket, inducing conformational changes that disrupt FUT8 activity. Leveraging these insights, we design CAIF to covalently target lysine K216 within the allosteric site. CAIF exhibits minimal cytotoxicity and significantly inhibits core fucosylation and cancer cell invasion in cellular assays. This work establishes CAIF as a lead compound for further optimization and development, offering a framework for targeting glycosyltransferases through allosteric and covalent inhibition strategies.

Covalent inhibitor design confers activity against both GDP- and GTP-bound forms of KRAS G12C

Matthew L. Condakes, Zhuo Zhang, Derek B. Danahy, Richard R. Moore, Sirish Kaushik Lakkaraju, Xiaoliang Zhuo, Yuka Amako, Robert M. Borzilleri, Srividya B. Balachander, Lisa Chourb, Rita L. Civiello, Ashok R. Dongre, Daniel P. Downes, Dieter M. Drexler, Brianne M. Dudiak, Liudmila Dzhekieva, Miriam El-Samin, Brian E. Fink, Kosea Frederick, Cherrie Huang, Javed Khan, Emma Lees, Christopher G. Levins, Courtney McCarthy, Michelle L. Stewart

Nat Commun (2026). 

https://doi.org/10.1038/s41467-026-69003-0

The discovery of KRAS G12C inactive state inhibitors represented a significant advancement in the field of precision oncology. While inactive state inhibition shows promise in patients, Switch II (SWII)-binding inhibitors targeting both inactive and active states remain an underdeveloped therapeutic modality. Here, we describe the discovery of such KRAS G12C dual inhibitors that bind the SWII allosteric site using a chemically differentiated warhead to covalently modify both the KRAS G12C inactive and active states. Co-crystal structures reveal that these inhibitors perturb a key water-mediated hydrogen bonding network and trigger allosteric remodeling of the GTP-bound protein surface and SWI that prevents binding to downstream effectors. Consistent with simultaneous targeting of the active and inactive states, dual inhibitors provide rapid covalent target engagement and suppression of MAPK signaling. However, they demonstrate similar efficacy in cellular and in vivo models when compared to inactive state-selective ones despite faster target inactivation. Furthermore, both inhibitor classes show similar cellular efficacy in the presence of growth factors that drive KRAS, wt NRAS, and wt HRAS to the active state. These data provide the first detailed account of targeting both the active and inactive states of KRAS G12C and highlight the absence of a mechanistic advantage in contexts dependent on prolonged target inhibition.

Development of a Lysine-Reactive Targeted Covalent Inhibitor (TCI) for the P300/CBP-Associated Factor (PCAF) Bromodomain Through Structure-Based Design

Richard Ede  and Kerstin E Peterson  and Richard Begyinah  and Irin P Tom  and Jason Ochoada  and Molly Sneddon  and Marcus Fischer  and Anang A Shelat  and William C K Pomerantz

ChemRxiv.  2026.

DOI: https://doi.org/10.26434/chemrxiv.10001717/v1

Epigenetics is defined by changes in heritable phenotypes that do not involve a change in DNA sequence. P300/CBP-associated factor (PCAF) is an important epigenetic regulatory protein that can alter chromatin through a histone acetyltransferase domain, while also serving as an epigenetic reader through a C-terminal bromodomain. PCAF promotes the transcription of the HIV-1 genome and is implicated in the development of glioblastoma. The currently reported PCAF inhibitors are non-covalent and require high concentration to maintain target occupancy. Here, we explore a new approach using covalent inhibition. Starting with a lead scaffold (BZ1), test-molecules were rationally designed for selectively targeting PCAF by installing lysine-reactive groups onto the lead scaffold to enable covalent bond formation with the non-conserved lysine residue in the PCAF bromodomain. The inhibition, selectivity, and kinetic properties (kinact/KI) of these molecules were evaluated using intact protein mass spectrometry, while biophysical, and cellular data were employed to verify covalent mechanism and in-cell target engagement. After optimization, we developed the first PCAF covalent inhibitor, 10, which labeled PCAF covalently in vitro and engages PCAF in cells. The covalent inhibitor, 10, represents a useful starting point for future inhibitor optimization and heterobifunctional molecule development.

Covalent Protein Inhibitors via Tyrosine and Tryptophan Conjugation with Cyclic Imine Mannich Electrophiles

Dr. Sijie Wang, Dr. Lei Wang, Dr. Marco Hadisurya, Dr. Siavash Shahbazi Nia, Prof. Dr. W. Andy Tao, Prof. Dr. Emily C. Dykhuizen, Prof. Dr. Casey J. Krusemark

Angewandte Chemie e16630

https://doi.org/10.1002/ange.202516630

Digital Object Identifier (DOI)

Targeted covalent inhibitors (TCIs) are increasingly popular as drug candidates and chemical probes. Among current TCIs, the chemistry is largely limited to cysteine and lysine side chain reactivity. Here, we investigated the utility of cyclic imine Mannich electrophiles as covalent warheads to target protein tyrosine and tryptophan side chains. We characterized the intrinsic reaction rates of several cyclic imines to tyrosine and other amino acid side chains and validated reactivity using protein affinity labeling of a cyclic imine-modified trimethoprim with tyrosine and tryptophan mutants of E. coli dihydrofolate reductase. To validate the utility of the approach, we appended cyclic imine warheads to a CBX8 chromodomain inhibitor to label a non-conserved tyrosine, which improved both the potency and selectivity of the inhibitor for CBX8 in vitro and in cells. These findings indicate that Mannich electrophiles are promising and robust chemical warheads for tyrosine and tryptophan bioconjugation and development of covalent inhibitors.

Chemoproteomics discovery of a CNS-penetrant covalent inhibitor of PIKfyve

Antony J. Burton, Louis S. Chupak, Alison J. Davis, Ahmed S. A. Mady, Mirco Meniconi, Barry Teobald, Bryan W. Dorsey, Lauren R. Byrne, Ryan Mulhern, Berent Lundeen, Elizabeth W. Sorensen, Bharti Patel, Sean Brennan, Dhiraj Kormocha, Ruben Tommasi, Graham L. Simpson, Jeffrey W. Keillor, Laura D’Agostino, Pearl S. Huang, Elayne Penebre

bioRxiv 2026.01.26.701341; 

doi: https://doi.org/10.64898/2026.01.26.701341

PIKfyve is a lipid kinase involved in regulating protein clearance mechanisms and is a promising target for the treatment of neurodegenerative diseases. Here, we present the discovery and optimization of a CNS-penetrant covalent PIKfyve inhibitor, DUN’058, which achieves sustained target occupancy in vivo. Covalent screening hits, identified from chemoproteomics experiments performed in live cells, were rapidly optimized to deliver a brain-penetrant covalent inhibitor of PIKfyve. This covalency centered approach employed a suite of mass spectrometry, biochemical and in vivo assays to optimize compound potency, selectivity, and CNS permeability. The target nucleophile, cysteine 1970, is on a flexible loop that appears distal from the kinase active site, highlighting the power of chemoproteomics screening to identify novel nucleophilic amino acids for covalent modification. DUN’058 achieves efficient covalency at the target cysteine, as well as highly selective covalent and reversible selectivity profiles. Covalent PIKfyve inhibition results in modulation of downstream pathway activity, including activation of the transcription factor TFEB, upregulation of protein clearance pathways, and increased GPNMB transcription and secretion of exosome markers. When dosed in vivo, DUN’058 achieves sustained target occupancy in the brains of mice long after systemic compound clearance, holding promise for achieving a sustained duration of action in the CNS at low doses, without prolonged effects in the periphery. Taken together, the development of DUN’058 is a demonstration of chemoproteomics-based discovery for a high value CNS target, providing an orally bioavailable and covalent PIKfyve inhibitor.