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.

Group Competition Strategy for Covalent Ligand Discovery

Zhihao Guo, Yunzhu Meng, Boyuan Zhao, Weidi Xiao, and Chu Wang

Journal of the American Chemical Society 2026

DOI: 10.1021/jacs.5c18150

As a powerful chemoproteomic tool, activity-based protein profiling (ABPP) has been extensively used for covalent ligand discovery. However, the current ABPP-based approaches are inherently based on indirect probe labeling competed by covalent ligands, and cannot directly compare the preferences of different ligands head-to-head. Herein, we report a group competition-based ABPP strategy (GC-ABPP) to allow the direct comparison of multiple ligands’ binding ability on a proteome-wide scale. By dividing a library of fully functionalized probes (FFPs) into different subgroups and labeling the proteome simultaneously, the direct competition enables comparison of the labeling ability of different probes in drawing a global protein–ligand affinity metric. When it is applied to an expanded probe library, this strategy can be used iteratively to select the highest-affinity ligand toward a certain target protein in a multiple-round process. As a proof of concept, we synthesized 65 FFPs and employed the GC-ABPP to screen the ligand–protein reactivity for >6000 cysteine sites. After three rounds of screening, we identified high-affinity ligands targeting BCAT2 and UGDH. Our “multiple ligands versus multiple proteins” screening paradigm demonstrates great potential for applications in covalent ligand/drug discovery.

Protein tyrosine phosphatase inactivation by electrophilic tyrosine modification

Madeleine L. Ware, David M. Leace, Zihan Qu, Quentin Schaefer, Sagar D. Vaidya, Mikayla L. Horvath, Zhihong Li, Yunpeng Bai, Zhong-Yin Zhang, and Ku-Lung Hsu 

Chem. Sci., 2026

https://doi.org/10.1039/D5SC07398G

Covalent protein tyrosine phosphatase (PTP) inhibitors principally target the catalytic cysteine, which is highly conserved and presents challenges for achieving selectivity across the PTP family. Here, we identified a tyrosine-reactive covalent inhibitor for SHP2 (DML189) with secondary molecular glue activity via a ligand induced protein tethering (LIPT) mechanism. We detected ligand binding at Y279, which is in proximity to the catalytic cysteine on SHP2 and has known functional and pathogenic properties. Covalent SHP2 modification by DML189 induced reversible disulfide tethering and monomer loss that was not observed to the same extent on PTP1B, LYP, or SHP1. Crosslinking mass spectrometry detected unique tethering events involving regulatory cysteines after DML189 modification on SHP2. Together, we discovered a tyrosine reactive inhibitor that targets functional sites on SHP2 and exhibits molecular glue activity through LIPT.

Covalent Peptide-Encoded Libraries Enable Discovery of Inhibitors of Epidermal Growth Factor Receptor (EGFR)

Ching-Pei Hsu, Michael Desgagné, Simon L. Rössler, Nathalie M. Grob, Charlotte E. Farquhar, Andrei Loas, Zena D. Jensvold, Hannah T. Baddock, Matthew Bratkowski, Aaron H. Nile, and Bradley Pentelute

ChemRxiv, 2026

doi:10.26434/chemrxiv-2026-z6vkt 

The use of encoding tags in combinatorial libraries accelerates hit generation by enabling high-throughput identification of small-molecule ligands. Peptide-encoded libraries (PELs) support the selection of structurally diverse small-molecule binders to proteins of interest. Here, we introduce a covalent PEL (coPEL) platform that incorporates cysteine-reactive scaffolds to identify irreversible protein binders. We leverage the chemical stability of PELs and the selective reactivity of palladium catalysts derived from dialkylbiaryl phosphine ligands to enable solid-phase Heck coupling reactions to rapidly diversify covalent acrylamide warheads. The optimized reaction conditions are high-yielding across a broad range of (hetero)aryl halides, ensuring robust performance and versatility within the coPEL platform. Screening a coPEL against the epidermal growth factor receptor (EGFR) tyrosine kinase, a key oncology target, yielded covalent small-molecule inhibitors with low-micromolar potency in vitro. This approach provides a complementary strategy for targeting diverse proteins and developing new classes of covalent inhibitors.